Insulin regulation of gene expression and concentrations of white adipose tissue-derived proteins in vivo in healthy men: relation to adiponutrin

in Journal of Endocrinology

Adiponutrin is a newly described white adipose tissue (WAT)-derived protein whose function and regulation remain widely unclear in humans though it is suggested to be related to insulin sensitivity. Recently, we found that adiponutrin expression is reduced in type 2 diabetic subjects in basal and insulin-stimulated states. To examine adiponutrin regulation by the insulin pathway in relation to other WAT-related proteins with well-known relation to insulin signaling and action, we examined in healthy young men (1) the association of adiponutrin with p85α PI3K and HKII, leptin, adiponectin, and acylation-stimulating protein (ASP) and (2) the regulation of adiponutrin and WAT-derived proteins by 3-h hyperinsulinemic euglycemic clamp (HIEG). At baseline (N = 20), adiponutrin expressions were positively correlated with those of p85α PI3K (R = 0.54, P = 0.017), HKII (R = 0.58, P = 0.010), and serum leptin (R = 0.51, P = 0.036), but not with any other parameter measured including insulin sensitivity. Hyperinsulinemia (N = 10, +2365% above baseline) significantly increased the expression of adiponutrin (+770%, P = 0.002), p85α PI3K (+150%, P = 0.033), HKII (+147%, P = 0.007), and serum leptin (+11%, P = 0.031), while it decreased serum adiponectin (−15%, P = 0.001). In the insulin-stimulated state, adiponutrin mRNA expression levels correlated with basal p85α PI3K (R = 0.76, P = 0.018) and HKII (R = 0.86, P = 0.003) expression levels, with percentage increase in insulin (R = 0.73, P = 0.040), and with insulin-stimulated state HKII (R = 0.82, P = 0.007), leptin (R = 0.84, P = 0.005), and adiponectin (R = 0.85, P = 0.004) mRNA levels. In healthy young men, adiponutrin expression is unregulated by hyperinsulinemia and is related to basal and/or insulin-stimulated p85α PI3K, HKII, adiponectin, and leptin expression levels. We hypothesize that insulin-mediated regulation of adiponutrin expression is under the PI3K pathway. The relevance of the present findings to reduced adiponutrin expression in type 2 diabetes is discussed.

Abstract

Adiponutrin is a newly described white adipose tissue (WAT)-derived protein whose function and regulation remain widely unclear in humans though it is suggested to be related to insulin sensitivity. Recently, we found that adiponutrin expression is reduced in type 2 diabetic subjects in basal and insulin-stimulated states. To examine adiponutrin regulation by the insulin pathway in relation to other WAT-related proteins with well-known relation to insulin signaling and action, we examined in healthy young men (1) the association of adiponutrin with p85α PI3K and HKII, leptin, adiponectin, and acylation-stimulating protein (ASP) and (2) the regulation of adiponutrin and WAT-derived proteins by 3-h hyperinsulinemic euglycemic clamp (HIEG). At baseline (N = 20), adiponutrin expressions were positively correlated with those of p85α PI3K (R = 0.54, P = 0.017), HKII (R = 0.58, P = 0.010), and serum leptin (R = 0.51, P = 0.036), but not with any other parameter measured including insulin sensitivity. Hyperinsulinemia (N = 10, +2365% above baseline) significantly increased the expression of adiponutrin (+770%, P = 0.002), p85α PI3K (+150%, P = 0.033), HKII (+147%, P = 0.007), and serum leptin (+11%, P = 0.031), while it decreased serum adiponectin (−15%, P = 0.001). In the insulin-stimulated state, adiponutrin mRNA expression levels correlated with basal p85α PI3K (R = 0.76, P = 0.018) and HKII (R = 0.86, P = 0.003) expression levels, with percentage increase in insulin (R = 0.73, P = 0.040), and with insulin-stimulated state HKII (R = 0.82, P = 0.007), leptin (R = 0.84, P = 0.005), and adiponectin (R = 0.85, P = 0.004) mRNA levels. In healthy young men, adiponutrin expression is unregulated by hyperinsulinemia and is related to basal and/or insulin-stimulated p85α PI3K, HKII, adiponectin, and leptin expression levels. We hypothesize that insulin-mediated regulation of adiponutrin expression is under the PI3K pathway. The relevance of the present findings to reduced adiponutrin expression in type 2 diabetes is discussed.

Keywords:

Introduction

Adiponutrin is a newly described non-secreted protein mainly expressed in brown and white adipose tissues (WAT; Baulande et al. 2001, Kershaw et al. 2006). To date, the physiological function of adiponutrin is unknown though it is suggested that adiponutrin may facilitate lipid storage in WAT (Jenkins et al. 2004, Lake et al. 2005, Kershaw et al. 2006). Similarly, the physiological regulation of adiponutrin expression is still being explored but several lines of evidence, mainly in rodents, suggest that adiponutrin may be regulated by energy balances and particularly by insulin.

In vitro, adiponutrin expression increases with differentiation of murine pre-adipocytes and is induced by glucose and insulin treatments of these cells (Baulande et al. 2001, Kershaw et al. 2006). Accordingly, adiponutrin expression in rodent’s WAT decreases with fasting (Baulande et al. 2001, Bertile & Raclot 2004, Wiesner et al. 2004, Kershaw et al. 2006) and increases with refeeding (Baulande et al. 2001, Kershaw et al. 2006) and with the ingestion of high-carbohydrate and high-protein meals (Polson & Thompson 2003, 2004). Moreover, WAT adiponutrin expression is decreased in two mice models of altered insulin action: the insulin-receptor knockout mice and the insulin-deficient mice (streptozotocin-induced diabetes), a defect that is corrected upon insulin replacement in this later mouse model (Kershaw et al. 2006). Finally, as adiponutrin expression is increased in obese fa/fa Zucker rats (having increased ob gene expression) (Guerre-Millo 1997, Baulande et al. 2001), while it is reduced in leptin-deficient obese ob/ob mice (Lake et al. 2005), leptin pathway may be implicated in the regulation of adiponutrin expression.

Little is known about adiponutrin regulation in human. To date only two studies were reported, results of which are partially inconsistent with each other and murine data. S.c. WAT adiponutrin expression was not increased in obese subjects and was associated with insulin sensitivity in one study (Liu et al. 2004) but not in the other (where insulin sensitivity was associated with adiponutrin expression in visceral WAT only; Johansson et al. 2006). However, both human studies suggest that adiponutrin expression may be regulated by insulin and glucose. In one study, adiponutrin expression in WATwas decreased after energy restriction and was increased upon refeeding (Liu et al. 2004) and in the other, adiponutrin expression in cultured human adipocytes was induced by insulin in a glucose-dependent manner (Johansson et al. 2006).

Clearly much remains to be explored regarding the regulation of adiponutrin in human, particularly given the possible role of adiponutrin in insulin sensitivity. Recently, we examined the regulation of adiponutrin expression by hyperinsulinemia in type 2 diabetic obese subjects. Despite their obese state and the origin of adiponutrin being WAT, fasting adiponutrin expression as well as insulin-stimulated increase in adiponutrin expression were reduced in obese diabetic subjects when compared with controls (Moldes et al. 2006).

Insulin sensitivity as well as the regulation of many WAT-derived proteins involved in insulin action are defective in type 2 diabetes and may affect the regulation of adiponutrin expression. To better understand the in vivo regulation of adiponutrin by the insulin pathway and explain its reduced expression in type 2 diabetes, we explored (1) the association of adiponutrin with insulin sensitivity and WAT-derived proteins of known relation to insulin signaling and action and (2) the concomitant regulation of adiponutrin with the WAT-derived proteins by insulin infusion. The WAT-derived proteins examined were hexokinase II (HK II), the regulatory subunit p85α of phosphatidylinositol-3 kinase (PI3K), leptin, adiponectin, and acylation-stimulating protein (ASP). These proteins have been well characterized and described in relation to insulin action in healthy and in reduced insulin sensitivity state-like type 2 diabetes (Lefai et al. 2001, Saltiel & Kahn 2001, Faraj et al. 2004).

Materials and Methods

Subjects and study design

Twenty lean, healthy Caucasian young men participated in the study. Subjects underwent routine medical examination and biological testing. None had familial or personal history of diabetes, obesity, dyslipidemia, or hypertension or was on any medication. All subjects were sedentary, having <2 h-structured exercise per week, and were instructed not to engage in any exercise for the previous 48 h before the study. The study was conducted after 12 h fast. All subjects gave written consent after being informed of the nature, purpose, and possible risks of the study. The experimental protocol was approved by the ethics committee of the University of Montreal.

Analytical procedures

Hyperinsulinemic euglycemic clamp (HIEG) technique

To examine the effect of hyperinsulinemia alone on adiponutrin expression in WAT in vivo, ten men underwent a 3-h HIEG clamp where hyperinsulinemia was coupled to euglycemia as previously described (DeFronzo et al. 1979). Briefly, plasma insulin was elevated to a plateau concentration using primed-constant exogenous insulin infusion at 75 mU/m2 per min. Plasma glucose was maintained at fasting levels using 20% dextrose infusion adjusted every 5–10 min according to repeated plasma glucose measurements. Insulin sensitivity measured by the clamp (Mclamp) represents glucose infusion rate at the last 30 min of the clamp at the steady state and is expressed as mg/kg per min (DeFronzo et al. 1979, Karelis et al. 2005). Insulin-stimulated data represent data analyzed at the last 30 min of the clamp.

Analysis of serum parameters

Fasting serum glucose concentrations were measured with a glucose analyzer (Beckman Glucose analyzer, Mississauga, ON, Canada). Serum triglyceride (TG) and total cholesterol were measured by an automated analyzer (Beckman-Coulter, Brea, CA, USA). Serum insulin, adiponectin, and leptin levels were measured in duplicate with a commercial RIA kit (Linco Research, St Charles, MO, USA) procedure using 125I-labeled bioactive human insulin, adiponectin, and leptin respectively as tracers and a rabbit polyclonal antibody raised against full-length peptides. Serum ASP was assayed by an in-house enzyme-linked immunosorbant assay using a monoclonal antibody as capture antibody and a polyclonal antibody as detecting antibody as described previously in detail (Maslowska et al. 1999). Serum leptin, adiponectin, and ASP were not measured for three subjects for lack of sufficient serum samples. Insulin sensitivity in the fasting state was calculated using quantitative insulin sensitivity check index (QUICKI) as 1/(log10 glucose+log10 insulin), where glucose is in mg/dl and insulin is in μU/ml (Rabasa-Lhoret et al. 2003).

White adipose tissue biopsy

S.c. abdominal WAT samples at the periumbilical level were obtained as previously described by needle biopsy under local anesthesia (Xylocaine 20 mg/ml, AstraZeneca; Vidal et al. 1996). Basal tissue samples were collected 1 h before the initiation of the clamp. Insulin-stimulated tissue samples were collected at the end of the 3 h clamp during which insulin infusion rate was maintained at 75 mU/m2 per min and plasma glucose concentration was maintained at fasting levels. Adipose tissues biopsies were immediately frozen in liquid nitrogen and stored at −80 °C for extraction of mRNA later.

Analysis of white adipose tissue mRNA expression levels

The WAT mRNA expression levels of adiponutrin, leptin, adiponectin, complement protein C3 (ASP precursor), p85α PI3K, and HKII were measured in the basal and the insulin-stimulated states. The total RNA was extracted from frozen WAT samples using the RNeasy Total RNA Minikit (Qiagen). First-strand cDNAs were synthesized from 250 ng total RNA in the presence of 100 U Superscript II (Invitrogen) using both random hexamers and oligo (dT) primers (Promega). Real-time PCR was performed using a LightCycler (Roche Diagnostics) in a final volume of 20 μl containing 5 μl of a 100-fold dilution of the reverse transcriptase (RT) reaction and 15 μl reaction buffer from the FastStart DNA Master SYBR Green kit (Roche Diagnostics) with 3 mM MgCl2 and the specific primers. After amplification, melting curve analysis was performed to verify the specificity of the reactions. For quantification, a standard curve was systematically generated with six different amounts (150–30 000 molecules/tube) of cDNA of the human target mRNA cloned in the pGEM plasmid (Promega). Analysis was performed using the LightCycler software (Roche Diagnostics). Gene-specific primers were designed to hybridize on different exons of the target human mRNA sequences accessible in the GenBank database.

The sequence of adiponutrin forward primer was 5′-GATGGAGGAGTGAGTGACAA-3′ and the reverse primer was 5′-CTGAATGCATCCAAATATCC-3′. The sequence of leptin forward primer was 5′-CAC-CAG-GAT-CAA-TGA-CAT-TTC-3′ and the reverse primer was 5′-TGC-CAG-TGT-CTG-GTC-CAT-CTT-G 3′. The sequence of adiponectin forward primer was 5′-AGG-CCG-TGA-TGG-CAG-AGA-TG-3′ and the reverse primer was 5′-CTT-CTC-CAG-GTT-CTC-CTT-TCC-TGC-3′. The sequence for C3 forward primer was 5′-GGA-TGA-GGA-CAT-CAT-TGC-AG-3′ and the reverse primer was 5′-GGC-TCT-TTC-AAG-TCC-TCA-AC-3′. The RT-quantitative PCR assays of these adipokines have been validated previously in human WAT for leptin mRNA (Vidal et al. 1996), adiponectin mRNA (Tan et al. 2005), and C3 mRNA (Dusserre et al. 2000). For HKII and p85α PI3K mRNA, the primers used for the RT-quantitative PCR have been published (Ducluzeau et al. 2001, Lefai et al. 2001). The expression levels of target genes were normalized according to a reference gene hypoxanthine phosphoribosyl-transferase(HPRT). Thesequence of HPRT forward primer was 5′-TTG-CTG-ACC-TGC-TGG-ATT-AC-3′ and the reverse primer was 5′-AGT-TGA-GAG-ATC-ATC-TCC-AC-3′.

Statistical analysis

Data are expressed as mean ± s.d. The correlation was analyzed by Pearson Product Moment correlation. In the correlation analysis with adiponutrin, one data point of high adiponutrin mRNA expression level was eliminated from both basal and insulin-stimulated states as they presented outliers/influential data that we believe created many false positives. The differences between subjects who did or did not undergo the HIEG clamp were examined with a two-tailed t-test, while the effect of insulin on the various measured serum and WAT parameters were analyzed by two-tailed paired t-test. Statistical analysis was performed using SigmaStat (Jandel, San Rafael, CA, USA) with significance set at P ≤ 0.05.

Results

Basal state

Basal anthropometric and metabolic characteristics of the 20 healthy, lean young men are presented in Table 1. As presented, adiponutrin is a relatively scarce protein as its fasting mRNA expression level was the least among the examined WAT-derived proteins, while adiponectin expression was the most abundant. At baseline, adiponutrin mRNA expression was positively correlated with the mRNA expression levels of p85α PI3K (R = 0.54, P = 0.017; Fig. 1) and HKII (R = 0.58, P = 0.010), and with serum leptin (R = 0.51, P = 0.036). There was no correlation between basal adiponutrin mRNA with any other WAT-derived protein or with age, weight, body mass index (BMI), serum lipids, insulin, glucose, or insulin sensitivity (QUICKI).

To better understand the networkof WAT signals from which adiponutrin is derived, we also examined the interaction of the measured WAT-derived proteins with each other. Serum adiponectin correlated with both leptin mRNA (R = 0.66, P = 0.004) and leptin concentration (R = 0.57, P = 0.016). The mRNA expression levels of p85α PI3K correlated with leptin concentration (R = 0.56, P = 0.019), but not with leptin mRNA or adiponectin and ASP concentration or mRNA. Moreover, the expression level of p85α PI3K mRNA correlated negatively with insulin (R = −0.51, P = 0.021) and positively with insulin sensitivity (QUICKI: R = 0.73, P<0.001), a pattern similar to that of HKII (insulin: R = −0.47, P = 0.038 and QUICKI: R = 0.62, P = 0.004). The expression levels of HKII mRNA and p85α PI3K mRNA were also highly correlated (R = 0.86, P<0.0001; Fig. 1). Similar to that with adiponutrin, basal insulin or QUICKI did not correlate with leptin, adiponectin, or C3 mRNA expression levels.

Insulin-stimulated state

Out of the 20 subjects examined in the basal state, ten underwent HIEG clamps. There was no difference in all measured characteristics between the subjects who did versus those who did not do the HIEG clamp except for lower TG and ASP concentrations in the clamp subjects (clamp subjects TG = 0.93 ± 0.19 mM versus non-clamp subjects TG = 1.33 ± 0.43 mM and clamp subjects ASP = 16.34 ± 7.86 nM versus non-clamp subjects ASP = 30.42 ± 5.98 nM, P<0.05 for TG and ASP).

The effect of HIEG clamp on the expression levels and the serum concentrations of the WAT-derived proteins examined in ten lean, healthy men is presented in Table 2 and Fig. 2. Considering the range of fasting plasma insulin in healthy subjects which is 36–179 pM (Pagana & Pagana 1997), the increase in serum insulin with the HIEG clamp was within physiological post-prandial concentrations (five to six times higher than that during fasting; English et al. 2003). The expression levels of the gene of reference HPRT were not affected by the HIEG clamp (Table 2). Hyperinsulinemia significantly increased serum concentrations of leptin and decreased that of adiponectin (Table 2), while it had no effect on the mRNA expression levels of these hormones (which may indicate an insulin effect at the post-transcription level only). In addition, hyperinsulinemia significantly increased the mRNA expression levels of adiponutrin, p85α PI3K, and HKII. There was no effect of 3 h hyperinsulinemia on the concentration of ASP or the expression levels of its precursor protein C3.

Insulin-stimulated adiponutrin remained correlated to basal p85α PI3K mRNA (R = 0.76, P = 0.018) and basal HKII mRNA (R = 0.86, P = 0.003), and was correlated with percentage increase in insulin during the clamp (R = 0.73, P = 0.040). The correlation of adiponutrin expression with the examined WAT-derived proteins in the insulin-stimulated state is presented in Fig. 3. Insulin-stimulated adiponutrin mRNA levels correlated with those of HKII (R = 0.82, P = 0.007), leptin (R = 0.84, P = 0.005), and adiponectin (R = 0.85, P = 0.004; Fig. 3). Insulin sensitivity measured by the clamp (Mclamp) did not correlate with any measured WAT-derived proteins, including adiponutrin, HKII, and p85α PI3K mRNA. Of note, despite high correlation of HKII and p85α PI3K mRNA at baseline, their mRNA expression levels were not correlated in the insulin-stimulated state.

Discussion

Recent findings by our group and others indicate that, despite being a WAT-derived protein, adiponutrin expression is not elevated in obese subjects (Liu et al. 2004, Moldes et al. 2006). Furthermore, fasting adiponutrin expression as well as insulin-stimulated increase in adiponutrin expression were reduced in obese type 2 diabetic subjects when compared with controls (Moldes et al. 2006). Therefore, the regulation of adiponutrin expression in human may be altered by reduced insulin sensitivity and altered regulation of WAT-derived proteins in relation to insulin signaling and action in states like type 2 diabetes. In this study, we examined the association of adiponutrin with insulin sensitivity and insulin-related WAT-derived proteins in both the basal and the insulin-stimulated states in healthy subjects to bypass the confounding effect of obesity and diabetes on the regulation of these WAT-derived proteins, including adiponutrin (Lefai et al. 2001, Johansson et al. 2006, Moldes et al. 2006).

The major findings of this study in healthy young men are that: (1) fasting adiponutrin expression level is correlated with that of p85α PI3K and HKII, and with serum leptin; (2) acute hyperinsulinemia simultaneously induces mRNA levels of adiponutrin, HKII, and PI3K, increases leptin concentration, and decreases adiponectin concentration; (3) insulin-stimulated adiponutrin expression is highly correlated to that of insulin-stimulated HKII, adiponectin, and leptin.

Novel findings in our study indicate that 3 h infusion of physiological doses of insulin induced an eightfold increase in adiponutrin expression, which was correlated to the percentage increase in insulin concentrations. Moreover, basal adiponutrin mRNA levels were highly correlated with those of p85α PI3K. PI3K has a pivotal role in the propagation of insulin signaling (Saltiel & Kahn 2001) and the regulatory subunit p85α examined in this study is the most abundant variant of the regulatory subunits of PI3K in human WAT (Lefai et al. 2001). Many genes involved in the insulin signaling and action are under the PI3K pathway, among which HKII is examined here (Pendergrass et al. 1998). In our study, adiponutrin expression profile mirrored that of HKII. Both adiponutrin and HKII expression levels were: (1) highly correlated to each other and to p85α PI3K in the basal state, (2) induced by insulin infusion, and (3) highly correlated in the insulin-stimulated state. Therefore, we hypothesize that, like HKII, insulin-stimulation of adiponutrin expression in human WAT may be under the PI3K pathway. Although in our study, adiponutrin expression did not correlate with that of p85α PI3K in the insulin-stimulated state, the same observation also applies to HKII, despite the previously documented regulation of HKII expression by the activity of PI3K pathway (Pendergrass et al. 1998).

Type 2 diabetic subjects have reduced insulin action, insulin-mediated upregulation of p85α PI3K in WAT (Ducluzeau et al. 2001), and expression of genes under the PI3K-dependent pathway like HKII (Pendergrass et al. 1998). Our hypothesis that insulin-mediated regulation of adiponutrin is under the PI3K-dependent pathway may therefore explain reduced adiponutrin expression in type 2 diabetes. However, it should be noted that the lack of correlation between adiponutrin expression and insulin sensitivity at fasting (QUICKI) and insulin-stimulated state (Mclamp), does not oppose our belief that the degree of insulin resistance may modulate adiponutrin expression. In the present study, the narrow ranges of insulin concentrations and sensitivity in our homogenous and healthy population may have hindered correlation analysis. However, type 2 diabetic subjects exhibit far less insulin sensitivity than our subjects which would drive reduced adiponutrin expression. Of note, the consistent correlation of adiponutrin expression to that of HKII, a major enzyme for the progress of glucose metabolism (Pendergrass et al. 1998) may also suggest that insulin-mediated adiponutrin expression may be glucose-dependent. In fact, this is supported by (1) our data that adiponutrin expression is only reduced in impaired insulin-sensitive and hyperglycemic (or glucose intolerant) type 2 diabetic subjects and not obese subjects (Moldes et al. 2006) and (2) in vitro findings in human adipocytes where insulin failed to induce adiponutrin expression at sub-physiological glucose concentrations (1 mM) as it did at physiological or supra-physiological concentrations of glucose (5 and 25 mM; Johansson et al. 2006). Therefore, insulin and glucose are both important regulators of adiponutrin expression.

Another novel finding in our study is that adiponutrin expression was correlated with leptin concentration and expression at the basal and the insulin-stimulated states respectively. Leptin is a WAT-derived factor that is documented to increase insulin sensitivity by various mechanisms in central and peripheral tissues (Faraj et al. 2004). Therefore, leptin pathway may either facilitate insulin regulation of adiponutrin or be directly involved in adiponutrin regulation. Supporting the later mechanism is the finding that adiponutrin expression is reduced in WAT of ob/ob mice, deficient for leptin gene (Lake et al. 2005), while is increased in fa/fa rats (Baulande et al. 2001), deficient for leptin receptor not leptin gene which is in fact increased in these rats (Guerre-Millo 1997). Despite hyperleptinemia in type 2 diabetes, leptin resistance is suggested to be a defect in these subjects (Zimmet et al. 1999). Accordingly, leptin resistance in type 2 diabetic would further explain our observation of a defective adiponutrin expression in these subjects (Moldes et al. 2006).

Similar to leptin, adiponectin increases the insulin sensitivity in peripheral tissue (Faraj et al. 2004). However, adiponectin is the only known WAT-derived protein whose expression and plasma concentration are consistently reduced in both murine and human obesity (Scherer et al. 1995, Arita et al. 1999, Statnick et al. 2000). Furthermore, in our study, hyperinsulinemia reduced adiponectin concentration as was documented by others (Yu et al. 2002). Adiponectin expression and concentration are reduced in obese type 2 diabetic subjects being driven by both their obesity and their chronic hyperinsulinemia states (Statnick et al. 2000). Therefore, high correlation of adiponutrin expression with that of adiponectin in the insulin-stimulated states would further predict reduced adiponutrin expression in obese type 2 diabetic subjects.

Adiponutrin mRNA levels in our study did not correlate with ASP concentrations or the expression of its precursor protein C3. Moreover, in contrast to adiponutrin, hyper-insulinemia did not affect ASP concentration or C3 expression in our study as was previously reported for C3 (Koistinen et al. 2001). Although ASP concentration and C3 expression are elevated in obese type 2 diabetic subjects (Koistinen et al. 2001, Yang et al. 2005), this was linked to their obesity rather than their insulin resistance in one study (Koistinen et al. 2001). Therefore, adiponutrin and ASP may be differently regulated in human. In fact, the production of ASP from human abdominal WAT increases with ingestion of a high-fat meal (Saleh et al. 1998) and chylomicrons are the most potent regulators of ASP production from human adipocytes (Maslowska et al. 1997, Scantlebury et al. 1998, 2001). On the contrary, adiponutrin expression in mice WAT is neither stimulated with high-fat meal (Polson & Thompson 2004) nor affected by treatments of cultured human adipocytes with fatty acids (Johansson et al. 2006). Therefore, ASP pathway and its regulators (lipid-related signals) are unlikely to contribute to the regulation of adiponutrin in human (Liu et al. 2004).

In conclusion, adiponutrin expression in s.c. WATof healthy, lean young men is strongly induced by acute hyperinsulinemia and is highly related to the expression levels of HKII and p85α PI3K. We hypothesize that like HKII, insulin-mediated regulation of adiponutrin expression in human WAT may be through a PI3K-dependent pathway. Accordingly, we believe that our recent finding of reduced adiponutrin expression in obese type 2 diabetic subjects may be driven by reduced insulin sensitivity, defective insulin-mediated regulation of PI3K pathway, and glucose metabolism (implying defective regulation of genes under the PI3K pathway like HKII), as well as an impaired leptin sensitivity and adiponectin expression in these subjects.

Table 1

Basal anthropometric and metabolic characteristics of the 20 lean young men

Mean ± s.d.Range
n = 20 for all presented parameters except serum leptin, adiponectin, and ASP (n = 17).
Basal characteristics (n = 20)
Age (years)23.1 ± 2.420.0–28.0
Weight (kg)69.0 ± 6.761.0–79.9
BMI (kg/m2)22.3 ± 1.519.8–24.1
Serum concentrations
    Glucose (mM)5.0 ± 0.44.2–5.4
    Insulin (pM)66.9 ± 30.616.5–159.2
    QUICKI0.35 ± 0.030.30–0.44
    Total cholesterol (mM)4.0 ± 0.82.6–5.1
    TG (mM)1.1 ± 0.40.6–2.1
    Leptin (ng/ml)2.8 ± 2.20.9–8.8
    Adiponectin (μg/ml)9.6 ± 3.75.2–20.6
    ASP (nM)25.1 ± 9.68.9–38.2
WAT mRNA expression levels
    HPRT1.6 ± 0.70.5–3.2
    Adiponutrin/HPRT0.3 ± 0.20.1–1.1
    Leptin/HPRT13.6 ± 11.44.4–24.3
    Adiponectin/HPRT83.4 ± 43.628.6–191.2
    C3/HPRT13.7 ± 4.74.4–24.3
    P85α PI3K/HPRT16.7 ± 14.64.7–65.5
    HKII/HPRT20.9 ± 17.94.9–75.1
Table 2

Effect of the HIEG clamp on the serum concentrations and mRNA expression levels of measured WAT-derived proteins in ten lean young men

BasalInsulin stimulatedPercentage changePvalue
n = 10 for all presented parameters except serum leptin, adiponectin, and ASP (n = 7).
Variables (n = 10)
Mclamp (mg glucose/kg/min)11.8 ± 1.8
Serum concentrations
    Glucose (mM)5.0 ± 0.65.2 ± 0.45.70.374
    Insulin (pM)67.5 ± 40.71102.1 ± 177.12364.6<0.001
    Leptin (ng/ml)3.8 ± 2.94.2 ± 3.210.80.031
    Adiponectin (μg/ml)11.6 ± 4.810.0 ± 4.5−15.00.001
    ASP (nM)16.3 ± 7.913.5 ± 13.5−15.00.304
WAT mRNA expression levels
    HPRT1.9 ± 0.72.1 ± 1.112.70.327
    Adiponutrin/HPRT0.3 ± 0.31.9 ± 1.2789.70.002
    Leptin/HPRT16.9 ± 13.120.6 ± 14.748.30.145
    Adiponectin/HPRT98.8 ± 47.8102.9 ± 54.96.30.744
    C3/HPRT12.8 ± 3.713.7 ± 4.26.40.086
    p85α PI3K/HPRT20.3 ± 17.239.1 ± 24.5150.20.033
    HKII/HPRT20.2 ± 16.841.8 ± 23.8146.70.007
Figure 1
Figure 1

Pearson correlation of WAT mRNA expression levels of p85α PI3K to those of adiponutrin (R = 0.54, P = 0.017) and HKII (R = 0.58, P = 0.010) in healthy, lean young men at baseline.

Citation: Journal of Endocrinology 191, 2; 10.1677/joe.1.06659

Figure 2
Figure 2

The expression levels of WAT-derived proteins at the basal and the insulin-stimulated states induced by HIEG clamps in healthy, lean young men. *P<0.05, P<0.01, and P<0.001.

Citation: Journal of Endocrinology 191, 2; 10.1677/joe.1.06659

Figure 3
Figure 3

Pearson correlation of adiponutrin with insulin sensitivity and WAT-derived proteins at the insulin-stimulated state induced by HIEG clamps in healthy, lean young men.

Citation: Journal of Endocrinology 191, 2; 10.1677/joe.1.06659

This study was funded by a grant from the Canadian Institute for Health Research to R R L (grant no. OHP-65393) and K C (grant no. OOP-69600). M F is supported by post-doctorate fellowship from CIHR and R R L by a fellowship from the Fonds de Recherche en santé du Québec (FRSQ). M M was supported by the Association pour la Recherche sur le Cancer (ARC) and an INSERM fellowship. K C is a research scholar of the FRSQ and holds a senior Canada Research Chair in Adipose Tissue. We acknowledge the help of David H St Pierre for technical assistance in the measurement of plasma leptin. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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  • DeFronzoRA Tobin JD & Andres R 1979 Glucose clamp technique: a method for quantifying insulin secretion and resistance. American Journal of Physiology – Endocrinology and Metabolism237E214–E223.

    • Search Google Scholar
    • Export Citation
  • DucluzeauPH Perretti N Laville M Andreelli F Vega N Riou JP & Vidal H 2001 Regulation by insulin of gene expression in human skeletal muscle and adipose tissue: evidence for specific defects in type 2 diabetes. Diabetes501134–1142.

    • Search Google Scholar
    • Export Citation
  • DusserreE Moulin P & Vidal H 2000 Differences in mRNA expression of the proteins secreted by the adipocytes in human subcutaneous and visceral adipose tissues. Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease150088–96.

    • Search Google Scholar
    • Export Citation
  • EnglishPJ Coughlin SR Hayden K Malik IA & Wilding JPH 2003 Plasma adiponectin increases postprandially in obese but not in lean subjects. Obesity Research11839–844.

    • Search Google Scholar
    • Export Citation
  • FarajM Lu HL & Cianflone K 2004 Diabetes lipids and adipocyte secretagogues. Biochemistry and Cell Biology82170–190.

  • Guerre-MilloM1997 Regulation of ob gene and overexpression in obesity. Biomedecine and Pharmacotherapy51318–323.

  • JenkinsCM Mancuso DJ Yan W Sims HF Gibson B & Gross RW 2004 Identification cloning expression and purification of three novel human calcium independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities. Journal of Biological Chemistry27948968–48975.

    • Search Google Scholar
    • Export Citation
  • JohanssonLE Hoffstedt J Parikh H Carlsson E Wabitsch M Bondeson AG Hedenbro J Tornqvist H Groop L & Ridderstrale M 2006 Variation in the adiponutrin gene influences its expression and associates with obesity. Diabetes55826–833.

    • Search Google Scholar
    • Export Citation
  • KarelisAD Faraj M Bastard JP St Pierre DH Brochu M Prud’homme D & Rabasa-Lhoret R 2005 The metabolically healthy but obese individual presents a favorable inflammation profile. Journal of Clinical Endocrinology Metabolism904145–4150.

    • Search Google Scholar
    • Export Citation
  • KershawEE Hamm JK Verhagen LAW Peroni O Katic M & Flier JS 2006 Adipose triglyceride lipase: function regulation by insulin and comparison with adiponutrin. Diabetes55148–157.

    • Search Google Scholar
    • Export Citation
  • KoistinenHA Vidal H Karonen SL Dusserre E Vallier P & Koivisto VA 2001 Plasma acylation stimulating protein concentration and subcutaneous adipose tissue C3 mRNA expression in nondiabetic and type 2 diabetic men. Arteriosclerosis Thrombosis and Vascular Biology211034–1039.

    • Search Google Scholar
    • Export Citation
  • LakeAC Sun Y Li JL Kim JE Johnson JW Li D Revett T Shih HH Liu W Paulsen JE et al.2005 Expression regulation and triglyceride hydrolase activity of adiponutrin family members. Journal of Lipid Research462477–2487.

    • Search Google Scholar
    • Export Citation
  • LefaiE Roques M Vega N Laville M & Vidal H 2001 Expression of the splice variants of the p85alpha regulatory subunit of phosphoinositide 3-kinase in muscle and adipose tissue of healthy subjects and type 2 diabetic patients. Biochemical Journal360117–126.

    • Search Google Scholar
    • Export Citation
  • LiuYM Moldes M Bastard JP Bruckert E Viguerie N Hainque B Basdevant A Langin D Pairault J & Clement K 2004 Adiponutrin: a new gene regulated by energy balance in human adipose tissue. Journal of Clinical Endocrinology and Metabolism892684–2689.

    • Search Google Scholar
    • Export Citation
  • MaslowskaM Scantlebury T Germinario R & Cianflone K 1997 Acute in vitro production of ASP in differentiated adipocytes. Journal of Lipid Research3821–31.

    • Search Google Scholar
    • Export Citation
  • MaslowskaM Vu H Phelis S Sniderman AD Rhode BM Blank D & Cianflone K 1999 Plasma acylation stimulating protein adipsin and lipids in non-obese and obese populations. European Journal of Clinical Investigation29679–686.

    • Search Google Scholar
    • Export Citation
  • MoldesM Beauregard G Faraj M Peretti N Ducluzeau PH Laville M Rabasa-Lhoret R Vidal H & Clément K 2006 Adiponutrin gene is regulated by insulin and glucose in human adipose tissue. European Journal of Endocrinology155461–468.

    • Search Google Scholar
    • Export Citation
  • PaganaKD & Pagana TJ 1997Diagnostic and Laboratory Test Reference. edn 3 Missouri USA: Mosby-Year Book Inc.

  • PendergrassM Koval J Vogt C Yki-Jarvinen H Iozzo P Pipek R Ardehali H Printz R Granner D DeFronzo RA et al.1998 Insulin-induced hexokinase II expression is reduced in obesity and NIDDM. Diabetes47387–394.

    • Search Google Scholar
    • Export Citation
  • PolsonDA & Thompson MP 2003 Adiponutrin mRNA expression in white adipose tissue is rapidly induced by meal-feeding a high-sucrose diet. Biochemical and Biophysical Research Communications301261–266.

    • Search Google Scholar
    • Export Citation
  • PolsonDA & Thompson MP 2004 Macronutrient composition of the diet differentially affects leptin and adiponutrin mRNA expression in response to meal feeding. Journal of Nutritional Biochemistry15242–246.

    • Search Google Scholar
    • Export Citation
  • Rabasa-LhoretR Bastard JP Jan V Ducluzeau PH Andreelli F Guebre F Bruzeau J Louche-Pellissier C MaItrepierre C Peyrat J et al.2003 Modified quantitative insulin sensitivity check index is better correlated to hyperinsulinemic glucose clamp than other fasting-based index of insulin sensitivity in different insulin-resistant states. Journal of Clinical Endocrinology Metabolism884917–4923.

    • Search Google Scholar
    • Export Citation
  • SalehJ Summers LKM Cianflone K Fielding BA Sniderman AD & Frayn KN 1998 Coordinated release of acylation stimulating protein (ASP) and triacylglycerol clearance by human adipose tissue in vivo in the postprandial period. Journal of Lipid Research39884–891.

    • Search Google Scholar
    • Export Citation
  • SaltielAR & Kahn R 2001 Insulin signalling and the regulation of glucose and lipid metabolism. Nature414799–806.

  • ScantleburyT Maslowska M & Cianflone K 1998 Chylomicron specific enhancement of acylation stimulating protein (ASP) and precursor protein C3 production in differentiated human adipocytes. Journal of Biological Chemistry27320903–20909.

    • Search Google Scholar
    • Export Citation
  • ScantleburyT Sniderman AD & Cianflone K 2001 Retinoic acid regulation of acylation stimulating protein (ASP) and complement C3 in human adipocytes. Biochemistry Journal356445–452.

    • Search Google Scholar
    • Export Citation
  • SchererPE Williams S Fogliano M Baldini G & Lodish HF 1995 A novel serum protein similar to C1q produced exclusively in adipocytes. Journal of Biological Chemistry27026746–26749.

    • Search Google Scholar
    • Export Citation
  • StatnickMA Beavers LS Conner LJ Corominola H Johnson D Hammond CD Rafaeloff-Phail R Seng T Suter TM Sluka JP et al.2000 Decreased expression of apM1 in omental and subcutaneous adipose tissue of humans with type 2 diabetes. International Journal of Experimental Diabetes Research181–88.

    • Search Google Scholar
    • Export Citation
  • TanGD Debard C Funahashi T Humphreys SM Matsuzawa Y Frayn KN Karpe F & Vidal H 2005 Changes in adiponectin receptor expression in muscle and adipose tissue of type 2 diabetic patients during rosiglitazone therapy. Diabetologia481585–1589.

    • Search Google Scholar
    • Export Citation
  • VidalH Auboeuf D De Vos P Staels B Riou JP Auwerx J & Laville M 1996 The expression of ob gene is not acutely regulated by insulin and fasting in human abdominal subcutaneous adipose tissue. Journal of Clinical Investigation98251–255.

    • Search Google Scholar
    • Export Citation
  • WiesnerG Morash BA Ur E & Wilkinson M 2004 Food restriction regulates adipose-specific cytokines in pituitary gland but not in hypothalamus. Journal of Endocrinology180R1–R6.

    • Search Google Scholar
    • Export Citation
  • YangY Lu HL Zhang J Yu HY Wang HW Zhang MX & Cianflone K 2005 Relationships among acylation stimulating protein adiponectin and complement C3 in lean vs obese type 2 diabetes. International Journal of Obesity30439–446.

    • Search Google Scholar
    • Export Citation
  • YuJG Javorschi S Hevener AL Kruszynska YT Norman RA Sinha M & Olefsky JM 2002 The effect of thiazolidinediones on plasma adiponectin levels in normal obese and type 2 diabetic subjects. Diabetes512968–2974.

    • Search Google Scholar
    • Export Citation
  • ZimmetP Boyko EJ Collier GR & de Courten M 1999 Etiology of the metabolic syndrome: potential role of insulin resistance leptin resistance and other players. Annals of the New York Academy of Sciences89225–44.

    • Search Google Scholar
    • Export Citation

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    Pearson correlation of WAT mRNA expression levels of p85α PI3K to those of adiponutrin (R = 0.54, P = 0.017) and HKII (R = 0.58, P = 0.010) in healthy, lean young men at baseline.

  • View in gallery

    The expression levels of WAT-derived proteins at the basal and the insulin-stimulated states induced by HIEG clamps in healthy, lean young men. *P<0.05, P<0.01, and P<0.001.

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    Pearson correlation of adiponutrin with insulin sensitivity and WAT-derived proteins at the insulin-stimulated state induced by HIEG clamps in healthy, lean young men.

  • AritaY Kihara S Ouchi N Takahashi M Maeda K Miyagawa J Hotta K Shimomura I Nakamura T Miyaoka K et al.1999 Paradoxical decrease of an adipose-specific protein adiponectin in obesity. Biochemical and Biophysical Research Communications25779–83.

    • Search Google Scholar
    • Export Citation
  • BaulandeS Lasnier F Lucas M & Pairault J 2001 Adiponutrin a transmembrane protein corresponding to a novel dietary- and obesity-linked mRNA specifically expressed in the adipose lineage. Journal of Biological Chemistry27633336–33344.

    • Search Google Scholar
    • Export Citation
  • BertileF & Raclot T 2004 Differences in mRNA expression of adipocyte-derived factors in response to fasting refeeding and leptin. Biochimica et Biophysica Acta1683101–109.

    • Search Google Scholar
    • Export Citation
  • DeFronzoRA Tobin JD & Andres R 1979 Glucose clamp technique: a method for quantifying insulin secretion and resistance. American Journal of Physiology – Endocrinology and Metabolism237E214–E223.

    • Search Google Scholar
    • Export Citation
  • DucluzeauPH Perretti N Laville M Andreelli F Vega N Riou JP & Vidal H 2001 Regulation by insulin of gene expression in human skeletal muscle and adipose tissue: evidence for specific defects in type 2 diabetes. Diabetes501134–1142.

    • Search Google Scholar
    • Export Citation
  • DusserreE Moulin P & Vidal H 2000 Differences in mRNA expression of the proteins secreted by the adipocytes in human subcutaneous and visceral adipose tissues. Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease150088–96.

    • Search Google Scholar
    • Export Citation
  • EnglishPJ Coughlin SR Hayden K Malik IA & Wilding JPH 2003 Plasma adiponectin increases postprandially in obese but not in lean subjects. Obesity Research11839–844.

    • Search Google Scholar
    • Export Citation
  • FarajM Lu HL & Cianflone K 2004 Diabetes lipids and adipocyte secretagogues. Biochemistry and Cell Biology82170–190.

  • Guerre-MilloM1997 Regulation of ob gene and overexpression in obesity. Biomedecine and Pharmacotherapy51318–323.

  • JenkinsCM Mancuso DJ Yan W Sims HF Gibson B & Gross RW 2004 Identification cloning expression and purification of three novel human calcium independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities. Journal of Biological Chemistry27948968–48975.

    • Search Google Scholar
    • Export Citation
  • JohanssonLE Hoffstedt J Parikh H Carlsson E Wabitsch M Bondeson AG Hedenbro J Tornqvist H Groop L & Ridderstrale M 2006 Variation in the adiponutrin gene influences its expression and associates with obesity. Diabetes55826–833.

    • Search Google Scholar
    • Export Citation
  • KarelisAD Faraj M Bastard JP St Pierre DH Brochu M Prud’homme D & Rabasa-Lhoret R 2005 The metabolically healthy but obese individual presents a favorable inflammation profile. Journal of Clinical Endocrinology Metabolism904145–4150.

    • Search Google Scholar
    • Export Citation
  • KershawEE Hamm JK Verhagen LAW Peroni O Katic M & Flier JS 2006 Adipose triglyceride lipase: function regulation by insulin and comparison with adiponutrin. Diabetes55148–157.

    • Search Google Scholar
    • Export Citation
  • KoistinenHA Vidal H Karonen SL Dusserre E Vallier P & Koivisto VA 2001 Plasma acylation stimulating protein concentration and subcutaneous adipose tissue C3 mRNA expression in nondiabetic and type 2 diabetic men. Arteriosclerosis Thrombosis and Vascular Biology211034–1039.

    • Search Google Scholar
    • Export Citation
  • LakeAC Sun Y Li JL Kim JE Johnson JW Li D Revett T Shih HH Liu W Paulsen JE et al.2005 Expression regulation and triglyceride hydrolase activity of adiponutrin family members. Journal of Lipid Research462477–2487.

    • Search Google Scholar
    • Export Citation
  • LefaiE Roques M Vega N Laville M & Vidal H 2001 Expression of the splice variants of the p85alpha regulatory subunit of phosphoinositide 3-kinase in muscle and adipose tissue of healthy subjects and type 2 diabetic patients. Biochemical Journal360117–126.

    • Search Google Scholar
    • Export Citation
  • LiuYM Moldes M Bastard JP Bruckert E Viguerie N Hainque B Basdevant A Langin D Pairault J & Clement K 2004 Adiponutrin: a new gene regulated by energy balance in human adipose tissue. Journal of Clinical Endocrinology and Metabolism892684–2689.

    • Search Google Scholar
    • Export Citation
  • MaslowskaM Scantlebury T Germinario R & Cianflone K 1997 Acute in vitro production of ASP in differentiated adipocytes. Journal of Lipid Research3821–31.

    • Search Google Scholar
    • Export Citation
  • MaslowskaM Vu H Phelis S Sniderman AD Rhode BM Blank D & Cianflone K 1999 Plasma acylation stimulating protein adipsin and lipids in non-obese and obese populations. European Journal of Clinical Investigation29679–686.

    • Search Google Scholar
    • Export Citation
  • MoldesM Beauregard G Faraj M Peretti N Ducluzeau PH Laville M Rabasa-Lhoret R Vidal H & Clément K 2006 Adiponutrin gene is regulated by insulin and glucose in human adipose tissue. European Journal of Endocrinology155461–468.

    • Search Google Scholar
    • Export Citation
  • PaganaKD & Pagana TJ 1997Diagnostic and Laboratory Test Reference. edn 3 Missouri USA: Mosby-Year Book Inc.

  • PendergrassM Koval J Vogt C Yki-Jarvinen H Iozzo P Pipek R Ardehali H Printz R Granner D DeFronzo RA et al.1998 Insulin-induced hexokinase II expression is reduced in obesity and NIDDM. Diabetes47387–394.

    • Search Google Scholar
    • Export Citation
  • PolsonDA & Thompson MP 2003 Adiponutrin mRNA expression in white adipose tissue is rapidly induced by meal-feeding a high-sucrose diet. Biochemical and Biophysical Research Communications301261–266.

    • Search Google Scholar
    • Export Citation
  • PolsonDA & Thompson MP 2004 Macronutrient composition of the diet differentially affects leptin and adiponutrin mRNA expression in response to meal feeding. Journal of Nutritional Biochemistry15242–246.

    • Search Google Scholar
    • Export Citation
  • Rabasa-LhoretR Bastard JP Jan V Ducluzeau PH Andreelli F Guebre F Bruzeau J Louche-Pellissier C MaItrepierre C Peyrat J et al.2003 Modified quantitative insulin sensitivity check index is better correlated to hyperinsulinemic glucose clamp than other fasting-based index of insulin sensitivity in different insulin-resistant states. Journal of Clinical Endocrinology Metabolism884917–4923.

    • Search Google Scholar
    • Export Citation
  • SalehJ Summers LKM Cianflone K Fielding BA Sniderman AD & Frayn KN 1998 Coordinated release of acylation stimulating protein (ASP) and triacylglycerol clearance by human adipose tissue in vivo in the postprandial period. Journal of Lipid Research39884–891.

    • Search Google Scholar
    • Export Citation
  • SaltielAR & Kahn R 2001 Insulin signalling and the regulation of glucose and lipid metabolism. Nature414799–806.

  • ScantleburyT Maslowska M & Cianflone K 1998 Chylomicron specific enhancement of acylation stimulating protein (ASP) and precursor protein C3 production in differentiated human adipocytes. Journal of Biological Chemistry27320903–20909.

    • Search Google Scholar
    • Export Citation
  • ScantleburyT Sniderman AD & Cianflone K 2001 Retinoic acid regulation of acylation stimulating protein (ASP) and complement C3 in human adipocytes. Biochemistry Journal356445–452.

    • Search Google Scholar
    • Export Citation
  • SchererPE Williams S Fogliano M Baldini G & Lodish HF 1995 A novel serum protein similar to C1q produced exclusively in adipocytes. Journal of Biological Chemistry27026746–26749.

    • Search Google Scholar
    • Export Citation
  • StatnickMA Beavers LS Conner LJ Corominola H Johnson D Hammond CD Rafaeloff-Phail R Seng T Suter TM Sluka JP et al.2000 Decreased expression of apM1 in omental and subcutaneous adipose tissue of humans with type 2 diabetes. International Journal of Experimental Diabetes Research181–88.

    • Search Google Scholar
    • Export Citation
  • TanGD Debard C Funahashi T Humphreys SM Matsuzawa Y Frayn KN Karpe F & Vidal H 2005 Changes in adiponectin receptor expression in muscle and adipose tissue of type 2 diabetic patients during rosiglitazone therapy. Diabetologia481585–1589.

    • Search Google Scholar
    • Export Citation
  • VidalH Auboeuf D De Vos P Staels B Riou JP Auwerx J & Laville M 1996 The expression of ob gene is not acutely regulated by insulin and fasting in human abdominal subcutaneous adipose tissue. Journal of Clinical Investigation98251–255.

    • Search Google Scholar
    • Export Citation
  • WiesnerG Morash BA Ur E & Wilkinson M 2004 Food restriction regulates adipose-specific cytokines in pituitary gland but not in hypothalamus. Journal of Endocrinology180R1–R6.

    • Search Google Scholar
    • Export Citation
  • YangY Lu HL Zhang J Yu HY Wang HW Zhang MX & Cianflone K 2005 Relationships among acylation stimulating protein adiponectin and complement C3 in lean vs obese type 2 diabetes. International Journal of Obesity30439–446.

    • Search Google Scholar
    • Export Citation
  • YuJG Javorschi S Hevener AL Kruszynska YT Norman RA Sinha M & Olefsky JM 2002 The effect of thiazolidinediones on plasma adiponectin levels in normal obese and type 2 diabetic subjects. Diabetes512968–2974.

    • Search Google Scholar
    • Export Citation
  • ZimmetP Boyko EJ Collier GR & de Courten M 1999 Etiology of the metabolic syndrome: potential role of insulin resistance leptin resistance and other players. Annals of the New York Academy of Sciences89225–44.

    • Search Google Scholar
    • Export Citation