Ghrelin- and GH-induced insulin resistance: no association with retinol-binding protein-4

Objective Supraphysiological levels of ghrelin and GH induce insulin resistance. Serum levels of retinol-binding protein-4 (RBP4) correlate inversely with insulin sensitivity in patients with type 2 diabetes. We aimed to determine whether ghrelin and GH affect RBP4 levels in human subjects. Materials and methods To study GH-independent effects of ghrelin, seven hypopituitary men undergoing replacement therapy with GH and hydrocortisone were given ghrelin (5 pmol/kg per min) and saline infusions for 300 min in a randomized, double-blind, placebo-controlled, crossover design. Circulating RBP4 levels were measured at baseline and during a hyperinsulinemic–euglycemic clamp on both study days. To study the direct effects of GH, nine healthy men were treated with GH (2 mg at 2200 h) and placebo for 8 days in a randomized, double-blind, placebo-controlled, crossover study. Serum RBP4 levels were measured before and after treatment, and insulin sensitivity was measured by the hyperinsulinemic–euglycemic clamp technique. Results Ghrelin acutely decreased peripheral insulin sensitivity. Serum RBP4 concentrations decreased in response to insulin infusion during the saline experiment (mg/l): 43.2±4.3 (baseline) vs 40.4±4.2 (clamp), P<0.001, but this effect was abrogated during ghrelin infusion (mg/l): 42.4±4.5 (baseline) vs 42.9±4.7 (clamp), P=0.73. In healthy subjects, serum RBP4 levels were not affected by GH administration (mg/l): 41.7±4.1 (GH) vs 43.8±4.6 (saline), P=0.09, although GH induced insulin resistance. Conclusions i) Serum RBP4 concentrations decrease in response to hyperinsulinemia, ii) ghrelin abrogates the inhibitory effect of insulin on circulating RBP4 concentrations, and iii) ghrelin as well as GH acutely induces insulin resistance in skeletal muscle without significant changes in circulating RBP4 levels.


Introduction
GH release from the pituitary gland is considered to be regulated at the hypothalamic level by GH releasing hormone (GHRH) and somatostatin (1). More recently, ghrelin, an endogenous ligand of the GH secretagogue receptor (GHS-R), has been identified in stomach tissue (2). When injected into the systemic circulation, ghrelin is a more potent releaser of GH compared with GHRH (3); in addition to this, ghrelin stimulates ACTH and prolactin secretion (4).
The observation that the GHS-R is also located in peripheral tissues indicates that ghrelin also exerts direct peripheral effects (5,6). It has recently been reported that exogenous ghrelin causes insulin resistance (7,8,9,10,11) and induces lipolysis (7,9,10,11,12,13). The diabetogenic effects of GH are well established (14,15), although the exact mechanisms by which GH induces insulin resistance remain to be elucidated.
Retinol-binding protein-4 (RBP4) is a protein and its serum levels are increased in patients with type 2 diabetes and correlate positively with several components of the metabolic syndrome (16). In rodent models, circulating RBP4 correlates positively with insulin resistance and RBP4 directly induces insulin resistance presumably by increased expression of the hepatic gluconeogenic enzyme phosphoenolpyruvate carboxykinase and impaired skeletal muscle insulin signaling (17). Moreover, genetic deletion or reduced serum concentrations of RBP4 by pharmacological remedies increase insulin sensitivity (17). A correlation between insulin sensitivity and serum concentrations of RBP4 has, however, not been consistently reported (18), and a number of reports have questioned the role of RBP4 in insulin resistance (19).
The aim of the present study was to investigate the potential effect of ghrelin-and GH-induced insulin resistance on circulating RBP4 concentrations. To study GH-independent effects of ghrelin, we conducted one experiment in adults with GH deficiency (GHD) receiving an acute i.v. ghrelin infusion; to study direct effects of GH, healthy subjects were treated with GH in a period of 8 days. Insulin sensitivity assessed by the euglycemic clamp technique and measurements of serum RBP4 levels were recorded.

Materials and methods
Subjects and experimental design Study 1 " Seven otherwise healthy hypopituitary men undergoing stable replacement therapy with GH and hydrocortisone (for O3 months) participated. The diagnosis of GHD was based on an insulin tolerance test (nZ6) or an arginine stimulation test (nZ1) with a GH cutoff level %3 mg/l. BMI was between 27.5 and 36.0 kg/m 2 and insulin-like growth factor 1 (IGF1) concentrations were within the normal range.
Each subject was examined twice in a randomized, double-blind, placebo (saline)-controlled, crossover manner. Each study day commenced at 0800 h in a quiet thermoneutral laboratory after 9 h of fasting. The subjects were examined in the supine position and allowed only to drink tap water. One i.v. cannula was placed in the antecubital region for infusions and one was placed in a contralateral dorsal hand vein for arterialized blood sampling.
Preparation of synthetic ghrelin: synthetic human acyl ghrelin (NeoMPS, Strasbourg, France) was dissolved in isotonic saline and sterilized by double passage through a 0.8/0.2 mm pore-size filter (Super Acrodisc, Gelman Sciences, Ann Arbor, MI, USA) by the local hospital pharmacy. From tZ0 to tZ300 min, the subjects received a primed continuous ghrelin infusion (5 pmol/kg per min) or a saline infusion. Three of the subjects had subcutaneous (periumbilical) adipose tissue biopsies by liposuction technique at tZ120 min after applying local analgesic of 10 ml lidocaine (xylocaine 10 mg/ml; AstraZeneca). A hyperinsulinemic-euglycemic clamp (plasma glucose clamped at 5.0 mmol/l, insulin 0.6 mU/kg per min, Actrapid, Novo Nordisk, Copenhagen, Denmark) was performed from tZ120 to tZ300 min. The period from tZ0 to tZ120 min is referred to as the basal period and the period from tZ120 to tZ300 min as the clamp period. Serum RBP4 levels were measured twice each study day: at baseline (tZK60 min) and during the clamp at tZ300 min. Data on glucose metabolism, insulin sensitivity, and energy expenditure from this study have previously been published in a report focusing on the direct metabolic effects of acute ghrelin infusion (11).
Each subject was examined immediately before and immediately after an 8-day treatment period with either GH (Norditropin SimplexX; Novo Nordisk; 2 mg s.c. at 1000 h, last injection on day 7) or placebo (saline) injections in a randomized, double-blind, crossover manner. The study periods were separated by a 1-to 3-week washout period.
Participants were prior to the study instructed by a clinical dietician to consume a weight-stable diet containing 50-60% carbohydrates, maximum 30% fat, and protein at 10-15% of total energy intake. Each study day commenced at 0800 h in a quiet, thermoneutral laboratory after an overnight fast. The subjects were examined in the supine position and were allowed only to drink tap water. One i.v. cannula was placed in the antecubital region for infusions and one was placed in a contralateral dorsal hand vein for arterialized blood sampling.
A hyperinsulinemic-euglycemic clamp (plasma glucose clamped at 5.0 mmol/l, insulin 0.6 mU/kg per min, Actrapid) was performed from tZ200 to tZ390 min. The period from tZ0 to tZ200 min is referred to as the 'basal period' and the period from tZ200 to tZ390 min as the 'clamp period.' Serum RBP4 was measured once each study day at tZ0.
Data on VLDL kinetics, i.m. triglyceride content, and insulin sensitivity have previously been published in a study focusing on the mechanisms by which GH cause lipolysis and insulin resistance (20).
Both studies were conducted in accordance with the Helsinki Declaration, and all subjects gave their oral and written informed consent to participate. The study protocols were approved by the Local Ethics Committee of Aarhus County, the Danish Medicines Agency and the Good Clinical Practice (GCP) Unit of Aarhus University Hospital.

Blood samples and measurements
Serum RBP4 levels were measured by an ELISA (EIA) from Alpco Diagnostics (Salem, NH, USA) with an intra-assay coefficient of variation (CV) of 5% and interassay CV of 9.7-9.8%. Plasma glucose was analyzed in duplicate using the glucose oxidase method (Beckman Instruments, Palo Alto, CA, USA). The levels of serum free fatty acid (FFA) were determined using a commercial kit (Wako Chemicals, Neuss, Germany). In study 1, insulin was analyzed with a double monoclonal immunofluorometric assay (Delfia, Perkin Elmer, Wallac Oy, Turku, Finland); in study 2, insulin was determined by commercial ELISA (DAKO, Glostrup, Denmark). IGF1 was measured by a Delfia in-house assay (21). Serum ghrelin (total levels) levels were measured in duplicate by an in-house assay as described previously (22). The assay measures immunoreactive levels of ghrelin using 125 I-labeled bioactive ghrelin tracer and rabbit polyclonal antibodies raised against octanoylated human ghrelin. The assay recognizes the COOH-terminal of ghrelin and as such determines acylated as well as des-acylated ghrelin. The intra-assay CV averaged 2.8% and samples from each individual were analyzed in one assay.

Isolation of RNA and real-time RT-PCR
Total RNA was isolated from adipose tissue using TRIzol (Gibco BRL, Life Technologies); RNA was quantified by measuring absorbance at 260 and 280 nm with a ratio R1.8 using a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Integrity of the RNA was checked by visual inspection of the two rRNAs, 18S and 28S, on an agarose gel. cDNA was synthesized with the Verso cDNA kit AB 1453 (Thermo Fisher Scientific, Inc.) using random hexamers. Real-time PCR for target gene was done with b2-microglobulin levels as internal control, and this expression did not change during intervention. The primers used for PCR are given in Table 1.
The PCRs were performed in duplicate using KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Inc., Woburn, MA, USA) in a LightCycler 480 (Roche Applied Science) using the following protocol: one step at 95 8C for 3 min, then 95 8C for 10 s, 60 8C for 20 s, and 72 8C for 10 s. The increase in fluorescence was measured in real time during the extension step. The relative gene expression was estimated using the default 'Advanced Relative Quantification' mode of the software version LCS 480 1.5.0.39 (Roche Applied Science).

Statistical analysis
Results are expressed as meanGS.E.M. or geometric mean and 25-75% range. Comparisons before and after treatment were carried out by a paired t-test or, if non-normal distributed data, with a signed rank test. Correlations were calculated by using Pearson's linear regression coefficient. A P value !0.05 was chosen as level of significance. The number of subjects is indicated by n. Analyses were performed using SigmaPlot version 11.0 for Windows.  Fig. 1).
Gene expressions " RBP4 levels in adipose tissue were increased during ghrelin administration as follows: subject 1, 27%; subject 2, 260%; and subject 5, 62% corresponding to an average increase of 117% in relation to the gene expression level during the saline experiment.
There was no correlation between insulin sensitivity and serum RBP4 concentrations after GH administration or after saline administration, PO0.05 (Fig. 5).

Discussion
Here, we report that insulin resistance induced by either ghrelin infusion in hypopituitary men or GH treatment in healthy young men is not associated with serum concentrations of RBP4. RBP4 is an adipokine reported to directly induce insulin resistance, whereas insulin sensitivity is improved by either genetic deletion of the RBP4 gene or lowering of serum concentrations of RBP4 (17). Yang et al. (17) first described that Glut4 K/K (Slc2a4 K/K ) insulinresistant mice had increased expression of Rbp4 gene in adipose tissue and that the PPARg agonist rosiglitazone, a well-known insulin sensitizer, reduced Rbp4 expression. They also described that skeletal muscle PI3K activity, considered to be a rate-limiting step in the insulin-signaling cascade, significantly decreases in the presence of excessive RBP4 levels in Rbp4 overexpression transgenic mice (17). So, in rodents, RBP4 is closely linked to insulin resistance. RBP4 is considered to be secreted from adipocytes in response to declining blood glucose levels and to cause insulin resistance and thereby counter regulate hypoglycemia. In support of this, RBP4 production is reported in human adipocytes (23) and RBP4 inhibits activation of IRS-1, a proximal enzyme in the insulin-signaling cascade, in vitro (24). In one clinical trial, serum RBP4 levels correlated with insulin resistance (in obese subjects) and impaired glucose tolerance or type 2 diabetes in nonobese subjects with a family disposition to type 2 diabetes (25). In healthy human subjects, data on the association between RBP4 levels and insulin sensitivity are divergent: Promintzer et al. (18) investigated whether there was an association between RBP4 levels and insulin sensitivity in healthy human subjects and they did not record any association. Ribel-Madsen et al.  used hyperinsulinemic-euglycemic clamps to investigate the association between RBP4 levels and insulin sensitivity. They reported that RBP4 correlated inversely with glucose rate of disappearance, but the association disappeared after adjusting for differences in adiponectin levels indicating that RBP4 is not a key regulator of peripheral glucose uptake. Kowalska et al. (27) investigated healthy lean and obese women with normal glucose tolerance and demonstrated an inverse correlation between RBP4 and glucose rate of disappearance independent of potential confounders. The reason for these discrepancies has not been established, but the potential association between circulating RBP4 and insulin sensitivity warrants further investigations and is the basis for our present study although the basis for expecting changes in circulating RBP4 by acute ghrelin or GH infusion is lacking currently.
Injection of ghrelin in human subjects elicits dosedependent GH and cortisol secretion (4), and infusion of ghrelin also increases plasma levels of glucose and FFAs (7,9,10,12) and induces peripheral insulin resistance (8,10). We have earlier investigated the GHS potency and diabetogenic effects of ghrelin infusions in healthy subjects without (9) and with concomitant somatostatin infusion (10) and reported that ghrelin induced GH secretion and insulin resistance in both settings. In the present study, we aimed to investigate the potential isolated effect of ghrelin and GH on serum RBP4 levels and therefore did not investigate the effect of ghrelin on RBP4 levels in healthy subjects. In the present study comprising hypopituitary subjects, ghrelin infusion did not result in GH or cortisol secretion (11). This demonstrates that the observed suppression of insulin-stimulated glucose disposal in skeletal muscle and increased FFA turnover were attributable to ghrelin infusion, whereas ghrelin did not affect hepatic insulin sensitivity (11) and RBP4 was not associated with ghrelin-induced insulin resistance. In a more recent report, we confirmed that ghrelin directly causes lipolysis in peripheral tissues (13). The mechanisms by which ghrelin induces insulin resistance and lipolysis remain to be investigated, and in this report, we have focused on the potential role of RBP4 in ghrelin-induced insulin resistance. Analyses of the adipose tissue by real-time PCR technique from the three patients, who volunteered to have fat biopsies taken, showed an association between ghrelin and RBP4 gene expression in adipose tissue. To investigate whether this association was due to a putative receptor-mediated effect of ghrelin on RBP4 gene expression in adipocytes, we performed a pilot study where we incubated human adipose tissueGghrelin for 48 h in vitro. We did not see any regulation of RBP4 mRNA under these circumstances, indicating that the increase in RBP4 gene expression in vivo could be an indirect effect of ghrelin. One suggestion is that the in vivo effect of ghrelin could be mediated through increased sympathetic nervous system activity (28), but more mechanistic studies are needed to fully elucidate this connection and this was not considered within the scope of the present study. Thus, acute ghrelin infusion upregulates RBP4 gene expression in adipose tissue, but this does not translate into measurable alterations in circulating RBP4 concentrations, suggesting that RBP4 is not involved in ghrelin-induced insulin resistance.
In study 1 comprising hypopituitary subjects, we recorded a significant reduction of serum RBP4 concentrations in response to hyperinsulinemia during saline administration, and this observation is in line with the observations by Promintzer et al. (18) who reported a decrease of plasma RBP4 in response to a hyperinsulinemic-euglycemic clamp in overweight healthy insulinsensitive as well as insulin-resistant adults. It has been hypothesized that decreased glucose uptake by adipocytes may stimulate RBP4 secretion as a counterregulatory response (17). If this mechanism is physiologically relevant, our observation could be explained by increased glucose uptake in adipocytes during hyperinsulinemia in the saline experiment and decreased glucose uptake in adipocytes during hyperinsulinemia in the ghrelin experiment, leading to i) a compensatory reduction of RBP4 secretion in the saline experiment and ii) no change in RBP4 secretion in the ghrelin experiment. Another hypothesis to explain this observation is that ghrelin infusion stimulates adipose tissue RBP4 gene expression (as indicated by the three adipose tissue biopsies) and RBP4 secretion, and this stimulating effect of ghrelin counteracts the inhibitory effect of insulin on serum RBP4 concentrations during saline infusion.
In the study comprising the healthy controls, insulin resistance was induced by pharmacological GH doses. The metabolic effects of GH have been recognized for decades (15) and GH signaling has recently been documented in human peripheral target tissues in vivo (29). The molecular mechanisms whereby GH induces insulin resistance in skeletal muscle, however, remain uncertain, and it is dubious whether they include distinct suppression of insulin signaling (30). In the present study, circulating RBP4 concentrations were not associated with GH-induced insulin resistance, suggesting that RBP4 does also not play a major role in GH-related changes of glucose homeostasis.
The strength of our two clinical studies is the crossover design where each subject is examined twice with and without ghrelin and GH respectively. The weakness of our study is the limited number of patients and healthy controls and, hence, the risk of making a type 2 error. There seems, however, not to be any effect of either ghrelin or GH on RBP4 levels at all. In both the ghrelin and the GH studies, insulin resistance was caused by reduced peripheral insulin sensitivity whereas insulin sensitivity of the liver remained unaffected. It is therefore likely that hepatic rather than peripheral insulin resistance interacts with RBP4.
In conclusion, these clinical studies do not support a causal association between insulin resistance and RBP4 serum levels in human subjects. The observed suppression of RBP4 levels in response to a hyperinsulinemiceuglycemic glucose clamp merits further investigation.
Declaration of interest M B Krag is a member of the advisory board on Diabetes and Dyslipidemia at Merck.

Study 1 was supported by a grant from the Danish Council for Independent
Research (Medical Sciences), an unrestricted grant from Novo Nordisk as well as grants from the Novo Nordisk Foundation, the A P Moller Foundation, the World Anti-Doping Agency, and the FOOD Study Group/-Ministry of Food, Agriculture, and Fisheries and Ministry of Family and Consumer Affairs. Microdialysis catheters were supplied by Roche. Study 2 was supported by Novo Nordisk who generously supplied GH preparations.
Author contribution statement E T Vestergaard was involved in the protocol, clinical trial, data analysis, discussion of results, manuscript first draft, and manuscript revision. M B Krag was involved in the protocol, clinical trial, data analysis, discussion of results, and manuscript revision. M M Poulsen, S B Pedersen, and N Jessen were involved in the PCR analyses, data analysis, discussion of results, and manuscript revision. N Moller and J O L Jorgensen were involved in the protocol, data analysis, discussion of results, and manuscript revision.