Robert V. Considine

Serum immunoreactive-leptin concentrations in normal-weight and obese humans.

BACKGROUND Leptin, the product of the ob gene, is a hormone secreted by adipocytes. Animals with mutations in the ob gene are obese and lose weight when given leptin, but little is known about the physiologic actions of leptin in humans. METHODS Using a newly developed radioimmunoassay, wer measured serum concentrations of leptin in 136 normal-weight subjects and 139 obese subjects (body-mass index, > or = 27.3 for men and > or = 27.8 for women; the body-mass index was defined as the weight in kilograms divided by the square of the height in meters). The measurements were repeated in seven obese subjects after weight loss and during maintenance of the lower weight. The ob messenger RNA (mRNA) content of adipocytes was determined in 27 normal-weight and 27 obese subjects. RESULTS The mean (+/- SD) serum leptin concentrations were 31.3 +/- 24.1 ng per milliliter in the obese subjects and 7.5 +/- 9.3 ng per milliliter in the normal-weight subjects (P < 0.001). There was a strong positive correlation between serum leptin concentrations and the percentage of body fat (r = 0.85, P < 0.001). The ob mRNA content of adipocytes was about twice as high in the obese subjects as in the normal-weight subjects (P < 0.001) and was correlated with the percentage of body fat (r = 0.68, P < 0.001) in the 54 subjects in whom it was measured. In the seven obese subjects studied after weight loss, both serum leptin concentrations and ob mRNA content of adipocytes declined, but these measures increased again during the maintenance of the lower weight. CONCLUSIONS Serum leptin concentrations are correlated with the percentage of body fat, suggesting that most obese persons are insensitive to endogenous leptin production.

Secretion of Angiogenic and Antiapoptotic Factors by Human Adipose Stromal Cells

Background—The delivery of autologous cells to increase angiogenesis is emerging as a treatment option for patients with cardiovascular disease but may be limited by the accessibility of sufficient cell numbers. The beneficial effects of delivered cells appear to be related to their pluripotency and ability to secrete growth factors. We examined nonadipocyte stromal cells from human subcutaneous fat as a novel source of therapeutic cells. Methods and Results—Adipose stromal cells (ASCs) were isolated from human subcutaneous adipose tissue and characterized by flow cytometry. ASCs secreted 1203±254 pg of vascular endothelial growth factor (VEGF) per 106 cells, 12 280±2944 pg of hepatocyte growth factor per 106 cells, and 1247±346 pg of transforming growth factor-&bgr; per 106 cells. When ASCs were cultured in hypoxic conditions, VEGF secretion increased 5-fold to 5980±1066 pg/106 cells (P =0.0016). The secretion of VEGF could also be augmented 200-fold by transfection of ASCs with a plasmid encoding VEGF (P <0.05). Conditioned media obtained from hypoxic ASCs significantly increased endothelial cell growth (P <0.001) and reduced endothelial cell apoptosis (P <0.05). Nude mice with ischemic hindlimbs demonstrated marked perfusion improvement when treated with human ASCs (P <0.05). Conclusions—Our experiments delineate the angiogenic and antiapoptotic potential of easily accessible subcutaneous adipose stromal cells by demonstrating the secretion of multiple potentially synergistic proangiogenic growth factors. These findings suggest that autologous delivery of either native or transduced subcutaneous ASCs, which are regulated by hypoxia, may be a novel therapeutic option to enhance angiogenesis or achieve cardiovascular protection.

Peroxisome proliferator-activated receptor gene expression in human tissues. Effects of obesity, weight loss, and regulation by insulin and glucocorticoids.

The peroxisome proliferator activated receptor (PPAR gamma) plays a key role in adipogenesis and adipocyte gene expression and is the receptor for the thiazolidinedione class of insulin-sensitizing drugs. The tissue expression and potential for regulation of human PPAR gamma gene expression in vivo are unknown. We have cloned a partial human PPAR gamma cDNA, and established an RNase protection assay that permits simultaneous measurements of both PPAR gamma1 and PPAR gamma2 splice variants. Both gamma1 and gamma2 mRNAs were abundantly expressed in adipose tissue. PPAR gamma1 was detected at lower levels in liver and heart, whereas both gamma1 and gamma2 mRNAs were expressed at low levels in skeletal muscle. To examine the hypothesis that obesity is associated with abnormal adipose tissue expression of PPAR gamma, we quantitated PPARgamma mRNA splice variants in subcutaneous adipose tissue of 14 lean and 24 obese subjects. Adipose expression of PPARgamma 2 mRNA was increased in human obesity (14.25 attomol PPAR gamma2/18S in obese females vs 9.9 in lean, P = 0.003). This increase was observed in both male and females. In contrast, no differences were observed in PPAR gamma1/18S mRNA expression. There was a strong positive correlation (r = 0.70, P < 0.001) between the ratio of PPAR gamma2/gamma1 and the body mass index of these patients. We also observed sexually dimorphic expression with increased expression of both PPAR gamma1 and PPAR gamma2 mRNAs in the subcutaneous adipose tissue of women compared with men. To determine the effect of weight loss on PPAR gamma mRNA expression, seven additional obese subjects were fed a low calorie diet (800 Kcal) until 10% weight loss was achieved. Mean expression of adipose PPAR gamma2 mRNA fell 25% (P = 0.0250 after a 10% reduction in body weight), but then increased to pretreatment levels after 4 wk of weight maintenance. Nutritional regulation of PPAR gamma1 was not seen. In vitro experiments revealed a synergistic effect of insulin and corticosteroids to induce PPAR gamma expression in isolated human adipocytes in culture. We conclude that: (a) human PPAR gamma mRNA expression is most abundant in adipose tissue, but lower level expression of both splice variants is seen in skeletal muscle; to an extent that is unlikely to be due to adipose contamination. (b) RNA derived from adipose tissue of obese humans has increased expression of PPAR gamma 2 mRNA, as well as an increased ratio of PPAR gamma2/gamma1 splice variants that is proportional to the BMI; (c) a low calorie diet specifically down-regulates the expression of PPAR gamma2 mRNA in adipose tissue of obese humans; (d) insulin and corticosteroids synergistically induce PPAR gamma mRNA after in vitro exposure to isolated human adipocytes; and (e) the in vivo modulation of PPAR gamma2 mRNA levels is an additional level of regulation for the control of adipocyte development and function, and could provide a molecular mechanism for alterations in adipocyte number and function in obesity.

Acute and chronic effects of insulin on leptin production in humans: Studies in vivo and in vitro.

This study was undertaken to investigate the changes in obesity (OB) gene expression and production of leptin in response to insulin in vitro and in vivo under euglycemic and hyperglycemic conditions in humans. Three protocols were used: 1) euglycemic clamp with insulin infusion rates at 40, 120, 300, and 1,200 mU · m−2 · min−1 carried out for up to 5 h performed in 16 normal lean individuals, 30 obese individuals, and 31 patients with NIDDM; 2) 64-to 72-h hyperglycemic (glucose 12.6 mmol/l) clamp performed on 5 lean individuals; 3) long-term (96-h) primary culture of isolated abdominal adipocytes in the presence and absence of 100 nmol/l insulin. Short-term hyperinsulinemia in the range of 80 to > 10,000 μU/ml had no effect on circulating levels of leptin. During the prolonged hyperglycemic clamp, a rise in leptin was observed during the last 24 h of the study (P < 0.001). In the presence of insulin in vitro, OB gene expression increased at 72 h (P < 0.01), followed by an increase in leptin released to the medium (P < 0.001). In summary, insulin does not stimulate leptin production acutely; however, a long-term effect of insulin on leptin production could be demonstrated both in vivo and in vitro. These data suggest that insulin regulates OB gene expression and leptin production indirectly, probably through its trophic effect on adipocytes.

Responses of Leptin to Short-Term Fasting and Refeeding in Humans: A Link With Ketogenesis but Not Ketones Themselves

We investigated the response of leptin to short-term fasting and refeeding in humans. A mild decline in subcutaneous adipocyte ob gene mRNA and a marked fall in serum leptin were observed after 36 and 60 h of fasting. The dynamics of the leptin decline and rise were further substantiated in a 6-day study consisting of a 36-h baseline period, followed by 36-h fast, and a subsequent refeeding with normal diet. Leptin began a steady decline from the baseline values after 12 h of fasting, reaching a nadir at 36 h. The subsequent restoration of normal food intake was associated with a prompt leptin rise and a return to baseline values 24 h later. When responses of leptin to fasting and refeeding were compared with that of glucose, insulin, fatty acids, and ketones, a reverse relationship between leptin and β-OH-butyrate was found. Consequently, we tested whether the reciprocal responses represented a causal relationship between leptin and β-OH-butyrate. Small amounts of infused glucose equal to the estimated contribution of gluconeogenesis, which was sufficient to prevent rise in ketogenesis, also prevented a fall in leptin. The infusion of β-OH-butyrate to produce hyperketonemia of the same magnitude as after a 36-h fast had no effect on leptin. The study indicates that one of the adaptive physiological responses to fasting is a fall in serum leptin. Although the mediator that brings about this effect remains unknown, it appears to be neither insulin nor ketones.

Evidence against either a premature stop codon or the absence of obese gene mRNA in human obesity.

Obese (ob) gene expression in abdominal subcutaneous adipocytes from lean and obese humans was examined. The full coding region of the ob gene was isolated from a human adipocyte cDNA library. Translation of the insert confirmed the reported amino acid sequence. There was no difference in the sequence of an reverse transcription PCR product of the coding region from five lean and five obese subjects. The nonsense mutation in the ob mouse which results in the conversion of arginine 105 to a stop codon was not present in human obesity. In all 10 human cDNAs, arginine 105 was encoded by CGG, consequently two nucleotide substitutions would be required to result in a stop codon. To compare the amount of ob gene expression in lean and obese individuals, radiolabed primer was used in the PCR reaction with beta-actin as a control. There was 72% more ob gene expression (P < 0.01) in eight obese subjects (body mass index, BMI = 42.8 +/- 2.7) compared to eight lean controls (BMI = 22.4 +/- 0.8). Regression analysis indicated a positive correlation between BMI and the amount of ob message (P < 0.005). There was no difference in the amount of beta-actin expression in the two groups. These results provide evidence that ob gene expression is increased in human obesity; furthermore, the mutations present in the mouse ob gene were not detected in the human mRNA population.

Synthesis of Leptin in Human Placenta

Gender-based differences in serum leptin levels have been reported in umbilical cord blood, and leptin has been detected in human amniotic fluid. In order to understand if leptin may be directly synthesized by human placentae an analysis made up of several steps was performed. First at all RT-PCR analysis from placenta-derived RNA was used to detect human leptin mRNA. The leptin-like immunoradioactivity detected in placentae extracts was identical to human leptin according to the criteria of charge, immunorecognition, SDS-PAGE analysis and blotting, indicating that intact leptin was found and no variants in size, charge or immunoactivity were present in the placentae. Finally an immunohistochemical analysis showed the presence of leptin in the cytoplasm of syncytiotrophoblast cells but not in the core of villi. In conclusion: leptin is synthesized as a single molecular variant identical to human recombinant leptin in human placentae at delivery.

Role of adiponectin in the protective action of dietary saturated fat against alcoholic fatty liver in mice

The protective effect of dietary saturated fatty acids against the development of alcoholic liver disease has long been known, but the underlying mechanism is not completely understood. We examined the involvement of the adipocyte hormone adiponectin. Circulating adiponectin levels were significantly elevated by chronic ethanol administration to mice consuming a diet high in saturated fat. The increase in circulating adiponectin was associated with the activation a set of hepatic signaling pathways mediated through AMP‐activated protein kinase, PPAR‐α, and PPAR‐γ coactivator α, which in turn led to markedly increased rates of fatty acid oxidation, prevention of hepatic steatosis, and alleviation of liver enzyme changes. Furthermore, treatment of rat 3T3‐L1 adipocytes with saturated fatty acids (palmitic or stearic acids) in the presence of ethanol increased secretion of adiponectin and enhanced activity of a mouse adiponectin promoter. In conclusion, the protective action of saturated fat against the development of alcoholic fatty liver in mice is partially mediated through induction of adiponectin. The present findings suggest a novel paradigm for dietary fatty acids in the pathogenesis of alcoholic liver disease and provide a promising therapeutic strategy—nutritional modulation of adiponectin—in treating human alcoholic fatty liver disease. (HEPATOLOGY 2005.)

Expression of the thermogenic nuclear hormone receptor coactivator PGC-1α is reduced in the adipose tissue of morbidly obese subjects

Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) is an accessory protein which can potentiate the transcriptional activation function of many nuclear hormone receptors. Its tissue distribution and physiological studies suggest that its principal in vivo roles are to promote cold-induced thermogenesis, mitochondrial biogenesis, hepatic gluconeogenesis, and fatty acid β-oxidation. It is expressed in the white adipose tissue of both humans and rodents, and in rodents it has been suggested to mediate in part the leptin-induced conversion of white adipocytes from fat storing to fat oxidising cells. In this study, quantitative real-time PCR has been used in human tissue to demonstrate that (1) PGC1α mRNA levels in subcutaneous fat are three-fold lower in morbidly obese than in slim subjects; (2) there are no differences in PGC1α mRNA between omental and subcutaneous mature adipocytes; (3) there is a robust induction of PGC1α expression during subcutaneous human preadipocyte differentiation ex vivo. Whether low PGC1α expression is a prelude to the development of obesity, or a consequence of that obesity, attempts to upregulate endogenous white adipose tissue expression may prove a valuable new avenue to explore in obesity therapy.

Viability of fat obtained by syringe suction lipectomy: effects of local anesthesia with lidocaine

The results of transplantation of free autologous fat obtained by blunt syringe suction lipectomy are unpredictable. We examined if adipose tissue viability is compromised by using syringe suction lipectomy and by infiltration of the tissue with local anesthetics. As reference, we used adipose tissue samples excised during elective surgery. Fat obtained intraoperatively and by lipectomy was digested with collagenase to isolate adipocytes. The mechanical damage associated with sample handling and cell isolation in both procedures was similar and did not exceed 6% of the total cell mass. In addition, cells isolated from intraoperative and lipectomy samples did not differ functionally, responded similarly to insulin stimulation of glucose transport and epinephrine-stimulated lipolysis, and retained the same growth pattern in culture. Since during fat transplantation the graft is exposed to local anesthetics at both the donor and the recipient sites, we reexamined adipocyte function in the presence of lidocaine. Lidocaine potently inhibited glucose transport and lipolysis in adipocytes and their growth in culture. That effect, however, persisted only as long as lidocaine was present; after washing, the cells were able to fully regain their function and growth regardless of whether the exposure was as short as 30 minutes or as long as 10 days. These results indicate that adipose tissue obtained by syringe lipectomy consists of fully viable and functional adipocytes, but local anesthetics may halt their metabolism and growth.