Publications

OBJECTIVE: Metreleptin has been efficacious in improving metabolic control in patients with lipodystrophy, but its efficacy has not been tested in obese patients with type 2 diabetes.

RESEARCH DESIGN AND METHODS: We studied the role of leptin in regulating the endocrine adaptation to long-term caloric deprivation and weight loss in obese diabetic subjects over 16 weeks in the context of a double-blinded, placebo-controlled, randomized trial. We then performed detailed interventional and mechanistic signaling studies in humans in vivo, ex vivo, and in vitro.

RESULTS: In obese patients with diabetes, metreleptin administration for 16 weeks did not alter body weight or circulating inflammatory markers but reduced HbA(1c) marginally (8.01 ± 0.93-7.96 ± 1.12, P = 0.03). Total leptin, leptin-binding protein, and antileptin antibody levels increased, limiting free leptin availability and resulting in circulating free leptin levels of ∼50 ng/mL. Consistent with clinical observations, all metreleptin signaling pathways studied in human adipose tissue and peripheral blood mononuclear cells were saturable at ∼50 ng/mL, with no major differences in timing or magnitude of leptin-activated STAT3 phosphorylation in tissues from male versus female or obese versus lean humans in vivo, ex vivo, or in vitro. We also observed for the first time that endoplasmic reticulum (ER) stress in human primary adipocytes inhibits leptin signaling.

CONCLUSIONS: In obese patients with diabetes, metreleptin administration did not alter body weight or circulating inflammatory markers but reduced HbA(1c) marginally. ER stress and the saturable nature of leptin signaling pathways play a key role in the development of leptin tolerance in obese patients with diabetes.

We examined the relationship between serum levels of leptin-binding protein (soluble leptin receptor [sOB-R]) and leptin with metabolic parameters at baseline and prospectively at 2-year follow-up in young healthy men. A total of 916 eighteen-year-old men were examined at baseline, with a subgroup of 91 participants examined again 2 years later. Anthropometric and metabolic measurements were performed at baseline and at follow-up. In the cross-sectional study, levels of sOB-R were significantly inversely correlated with all baseline measures of obesity and metabolic risk factors (blood pressure, total and low-density lipoprotein cholesterol, and fasting glucose), and significantly positively correlated with high-density lipoprotein cholesterol. After correcting for age, smoking status, and waist-to-hip ratio, the inverse correlation remained statistically significant for all measures of adiposity, fasting glucose, and the metabolic syndrome score. Correlations for leptin were similar in magnitude but opposite in direction to correlations for sOB-R. In prospective analyses, baseline levels of sOB-R were predictive at 2-year follow-up of fasting glucose, the metabolic syndrome score, and measures of adiposity in both unadjusted and adjusted models. Similarly, leptin was predictive of fasting glucose, the metabolic syndrome score, adiposity, and systolic blood pressure. We confirm correlations of leptin and sOB-R levels with measures of adiposity and metabolic risk factors at baseline, and demonstrate for the first time prospectively the role of sOB-R as an independent, although weak, predictor of metabolic syndrome and fasting glucose in young men.

OBJECTIVE: Short-term energy deprivation reduces leptin concentrations and alters the levels of circulating hormones of the hypothalamic-pituitary-peripheral axis in lean subjects. Whether the reduction in leptin concentration during long-term weight loss in obese individuals is linked to the same neuroendocrine changes seen in lean, leptin-sensitive subjects remains to be fully clarified.

METHODS: In this study, 24 overweight and obese adults (16 women and eight men; body mass index (BMI): 27.5-38.0 kg/m(2)) were prescribed a hypocaloric diet (-500 kcal/day) and were randomized to receive recombinant methionyl leptin (n=18, metreleptin, 10 mg/day self-injected s.c.) or placebo (n=6, same volume and time as metreleptin) for 6 months.

RESULTS: Metreleptin administration did not affect weight loss beyond that induced by hypocaloric diet alone (P for interaction=0.341) but increased the serum concentrations of total leptin by six- to eight-fold (P<0.001) and led to the generation of anti-leptin antibodies. Despite free leptin concentration (P for interaction=0.041) increasing from 9±1 ng/ml at baseline to 43±15 and 36±12 ng/ml at 3 and 6 months, respectively, changes in circulating hormones of the thyroid and IGF axes at 3 and 6 months were not significantly different in the placebo- and metreleptin-treated groups.

CONCLUSIONS: Leptin does not likely mediate changes in neuroendocrine function in response to weight loss induced by a mild hypocaloric diet in overweight and obese subjects.

OBJECTIVE: Lipocalin 2 (LCN2 or NGAL), a protein derived from neutrophils, macrophages, adipocytes, and other cells, has been proposed to be a link between obesity and insulin resistance (IR), but animal and cross-sectional human studies have revealed conflicting results. We studied the association of serum lipocalin 2 with anthropometric, metabolic, and cardiovascular risk markers in young healthy men cross-sectionally and, for the first time, prospectively after 2 years of follow-up, with and without adjustment for potential confounders including serum creatinine.

DESIGN: Two hundred and seventy-two participants were randomly selected from the Cyprus Metabolism Study (1056 men, 18 years), of whom 93 subjects participated in the follow-up study 2 years after baseline assessment. Associations were also explored between total and free leptin levels (to serve as positive controls) and anthropometric metabolic variables.

RESULTS: In the cross-sectional study, lipocalin 2 levels were marginally correlated in the unadjusted model with central fat distribution but not with body weight or total body fat mass. After adjusting for age, smoking, activity, body mass index, fat percentage, waist-to-hip ratio, and serum creatinine, no correlation was found with any cardiovascular risk factor. There was no correlation with the homeostasis model assessment of IR (HOMA-IR) at baseline. In the prospective analyses, baseline levels of lipocalin 2 were not predictive of any variables in unadjusted or adjusted models. As expected, total and free leptin were associated with anthropometric and metabolic variables both cross-sectionally and prospectively.

CONCLUSIONS: We demonstrate that lipocalin 2 is not an independent predictor of metabolic and cardiovascular risk factors in young men cross-sectionally or prospectively.

CONTEXT: Circulating adiponectin has been inversely associated with risk for several malignancies. Its association with thyroid cancer has not yet been evaluated.

OBJECTIVE/METHODS: We measured circulating adiponectin levels in 175 thyroid carcinoma patients and 107 controls. We also examined the expression of adiponectin receptors (AdipoR1 and AdipoR2) using immunohistochemistry in 82 thyroid carcinoma tissues and using RT-qPCR in 40 human thyroid carcinoma tissues (32 papillary, six follicular/Hurthle, one anaplastic, one medullary), four normal human thyroid tissue specimens, and the BHP7 and SW579 thyroid cancer cell lines. We then utilized these thyroid cancer cell lines to investigate whether adiponectin could directly regulate cell cycle or apoptosis.

RESULTS: Thyroid cancer patients had lower circulating adiponectin levels than controls (17.00 ± 6.32 vs. 19.26 ± 6.28 μg/ml; P < 0.001). Subjects in the highest tertile of circulating adiponectin concentrations had significantly lower odds of developing any type of thyroid carcinoma (odds ratio = 0.29; 95% confidence interval, 0.16-0.55), or papillary thyroid carcinoma (odds ratio = 0.27; 95% confidence interval, 0.14-0.55), before and after adjustment for potential confounders. Both thyroid carcinoma cell lines and tissues expressed AdipoR1 and AdipoR2. Recombinant adiponectin did not exert a clinically significant direct effect on cell cycle, proliferation, or apoptosis in thyroid cancer cell lines in vitro.

CONCLUSIONS: Circulating adiponectin is independently and inversely associated with the risk of thyroid cancer. Human thyroid carcinomas and cell lines express adiponectin receptors. However, in the absence of a major direct effect of adiponectin on thyroid cancer cell lines in vitro, the negative association observed herein may be attributed to the metabolic effects of adiponectin.

Obesity and diabetes have been associated with increased consumption of highly processed foods, and reduced consumption of whole grains and nuts. It has been proposed, mainly on the basis of observational studies, that nuts may provide superior satiation, may lead to reduced calorie consumption, and may decrease the risk of type 2 diabetes; but evidence from randomized, interventional studies is lacking. A total of 20 men and women with the metabolic syndrome participated in a randomized, double-blind, crossover study of walnut consumption. Subjects had two 4-day admissions to the clinical research center where they were fed an isocaloric diet. In addition, they consumed shakes for breakfast containing either walnuts or placebo (shakes were standardized for calories, carbohydrate, and fat content). Appetite, insulin resistance, and metabolic parameters were measured. We found an increased level of satiety (overall P value = 0.0079) and sense of fullness (P = 0.05) in prelunch questionnaires following the walnut breakfast as compared to the placebo breakfast, with the walnut effect achieving significance on day 3 and 4 (P = 0.02 and P = 0.03). We did not find any change in resting energy expenditure, hormones known to mediate satiety, or insulin resistance when comparing the walnut vs. placebo diet. Walnut consumption over 4 days increased satiety by day 3. Long-term studies are needed to confirm the physiologic role of walnuts, the duration of time needed for these effects to occur, and to elucidate the underlying mechanisms.

Adiponectin has been proposed to be a mediator of obesity-associated malignancies and to have direct antineoplastic effects acting via adiponectin receptors AdipoR1 and AdipoR2. We describe herein the expression of AdipoR1 and AdipoR2 in several cancers not previously studied. We used immunohistochemistry to assess expression of adiponectin receptors in archival specimens of renal cell carcinoma (n = 64), hepatocellular carcinoma (n = 123), melanoma (n = 20), cholangiocarcinoma (n = 20), transitional cell carcinoma of the bladder (n = 24), ovarian epithelial carcinoma (n = 63), cervical squamous cell carcinoma (n = 49), and adrenocortical carcinoma (n = 48). To compare expression in malignant versus nonmalignant tissues, we also studied AdipoR1 and AdipoR2 expression in pairs of renal cell carcinoma and adjacent healthy kidney tissue specimens by immunohistochemistry. We also studied mRNA expression in 45 specimens of renal cell carcinoma by real-time polymerase chain reaction. Finally, we utilized Western blotting to confirm the presence of adiponectin receptors and subsequently studied cell signaling pathways of adiponectin in the renal cancer cell line 786-O. Cancers associated with obesity were significantly more likely to express AdipoR1 than cancers not associated with obesity. Of the specimens of renal cell carcinoma, which is strongly associated with obesity, 93.8% expressed AdipoR1 compared to 44.9% of the specimens of cervical cell carcinoma, which is not associated with obesity (p < 0.001). There was no difference in the expression of adiponectin receptors or their mRNA between malignant and benign kidney tissue specimens. Overall, there were no correlations between expression of adiponectin receptors or their mRNA and tumor prognostic factors. Finally, Western blotting confirmed the presence of AdipoR1 in the renal cancer cell line 786-O, and adiponectin activates in vitro several signaling pathways in this cell line. In summary, we report for the first time expression of AdipoR1 and AdipoR2 in the above cancers and that AdipoR1 is more ubiquitously expressed in obesity-associated cancers.

Glucagon-like peptide-2 (GLP-2) is a nutrient-regulated intestinotrophic hormone derived from proglucagon in the distal intestine. Enteral nutrients (EN) potentiate the action of GLP-2 to reverse parenteral nutrition (PN)-induced mucosal hypoplasia. The objective was to determine what enteral protein component, casein, soy, or whey protein, potentiates the intestinal growth response to GLP-2 in rats with PN-induced mucosal hypoplasia. Rats received PN and continuous intravenous infusion of GLP-2 (100 microg/kg/day) for 7 days. Six EN groups received PN+GLP-2 for days 1-3 and partial PN+GLP-2 plus EN for days 4-7. EN was provided by ad libitum intake of a semielemental liquid diet with different protein sources: casein, hydrolyzed soy, whey protein concentrate (WPC), and hydrolyzed WPC+casein. Controls received PN+GLP-2 alone. EN induced significantly greater jejunal sucrase activity and gain of body weight, and improved feed efficiency compared with PN+GLP-2 alone. EN induced greater ileal proglucagon expression, increased plasma concentration of bioactive GLP-2 by 35%, and reduced plasma dipeptidyl peptidase IV (DPP-IV) activity compared with PN+GLP-2 alone, P < 0.05. However, only whey protein, and not casein or soy, potentiated the ability of GLP-2 to reverse PN-induced mucosal hypoplasia and further increase ileal villus height, crypt depth, and mucosa cellularity compared with PN+GLP-2 alone, P < 0.05. The ability of whey protein to induce greater mucosal surface area was associated with decreased DPP-IV activity in ileum and colon compared with casein, soy, or PN+GLP-2 alone, P < 0.05. In conclusion, whey protein potentiates the action of GLP-2 to reverse PN-induced mucosal hypoplasia in association with decreased intestinal DPP-IV activity.

BACKGROUND: Bowel resection may lead to short bowel syndrome (SBS), which often requires parenteral nutrition (PN) due to inadequate intestinal adaptation. The objective of this study was to determine the time course of adaptation and proglucagon system responses after bowel resection in a PN-dependent rat model of SBS.

METHODS: Rats underwent jugular catheter placement and a 60% jejunoileal resection + cecectomy with jejunoileal anastomosis or transection control surgery. Rats were maintained exclusively with PN and killed at 4 hours to 12 days. A nonsurgical group served as baseline. Bowel growth and digestive capacity were assessed by mucosal mass, protein, DNA, histology, and sucrase activity. Plasma insulin-like growth factor I (IGF-I) and bioactive glucagon-like peptide 2 (GLP-2) were measured by radioimmunoassay.

RESULTS: Jejunum cellularity changed significantly over time with resection but not transection, peaking at days 3-4 and declining by day 12. Jejunum sucrase-specific activity decreased significantly with time after resection and transection. Colon crypt depth increased over time with resection but not transection, peaking at days 7-12. Plasma bioactive GLP-2 and colon proglucagon levels peaked from days 4-7 after resection and then approached baseline. Plasma IGF-I increased with resection through day 12. Jejunum and colon GLP-2 receptor RNAs peaked by day 1 and then declined below baseline.

CONCLUSIONS: After bowel resection resulting in SBS in the rat, peak proglucagon, plasma GLP-2, and GLP-2 receptor levels are insufficient to promote jejunal adaptation. The colon adapts with resection, expresses proglucagon, and should be preserved when possible in massive intestinal resection.

Luminal nutrients stimulate structural and functional regeneration in the intestine through mechanisms thought to involve insulin-like growth factor I (IGF-I) and glucagon-like peptide-2 (GLP-2). We investigated the relationship between IGF-I and GLP-2 responses and mucosal growth in rats fasted for 48 h and then refed for 2 or 4 days by continuous intravenous or intragastric infusion or ad libitum feeding. Fasting induced significant decreases in body weight, plasma concentrations of IGF-I and bioactive GLP-2, jejunal mucosal cellularity (mass, protein, DNA, and villus height), IGF-I mRNA, and ileal proglucagon mRNA. Plasma IGF-I concentration was restored to fed levels with 2 days of ad libitum refeeding but not with 4 days of intravenous or intragastric refeeding. Administration of an inhibitor of endogenous GLP-2 (rat GLP-2 3-33) during ad libitum refeeding partially attenuated mucosal growth and prevented the increase in plasma IGF-I to fed levels; however, plasma GLP-2 and jejunal IGF-I mRNA were restored to fed levels. Intragastric refeeding restored intestinal cellularity and functional capacity (sucrase activity and sodium-glucose transporter-1 expression) to fed levels, whereas intravenous refeeding had no effect. Intestinal regeneration after 4 days of intragastric or 2 days of ad libitum refeeding was positively associated with increases in plasma concentrations of GLP-2 and jejunal IGF-I mRNA. These data suggest that luminal nutrients stimulate intestinal growth, in part, by increased expression of both GLP-2 and IGF-I.