Jerzy W. Kolaczynski

Decreased cerebrospinal-fluid/serum leptin ratio in obesity: a possible mechanism for leptin resistance

BACKGROUND A receptor for leptin has been cloned from the choroid plexus, the site of cerebrospinal-fluid (CSF) production and the location of the blood/cerebrospinal-fluid barrier. Thus, this receptor might serve as a transporter for leptin. We have studied leptin concentrations in serum and (CSF). METHODS AND FINDINGS We demonstrated by radioimmunoassay and western blot the presence of leptin in human CSF. We then measured leptin in CSF and serum in 31 individuals with a wide range of bodyweight. Mean serum leptin was 318% higher in 8 obese (40.2 [SE 8.6] ng/mL) than in 23 lean individuals (9.6 [1.5] ng/mL, p < 0.0005). However, the CSF leptin concentration in obese individuals (0.337 [0.04] ng/mL) was only 30% higher than in lean people (0.259 [0.26] ng/mL, p < 0.1). Consequently, the leptin CSF/serum ratio in lean individuals (0.047 [0.010]) was 4.3-fold higher than that in obese individuals (0.011 [0.002], p < 0.05). The relation between CSF leptin and serum leptin was best described by a logarithmic function (r = 0 x 52, p < 0.01). INTERPRETATION Our data suggest that leptin enters the brain by a saturable transport system. The capacity of leptin transport is lower in obese individuals, and may provide a mechanism for leptin resistance.

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.

Evidence of free and bound leptin in human circulation. Studies in lean and obese subjects and during short-term fasting.

Little is known about leptins interaction with other circulating proteins which could be important for its biological effects. Sephadex G-100 gel filtration elution profiles of 125I-leptin-serum complex demonstrated 125I-leptin eluting in significant proportion associated with macromolecules. The 125I-leptin binding to circulating macromolecules was specific, reversible, and displaceable with unlabeled leptin (ED50: 0.73 +/- 0.09 nM, mean +/- SEM, n = 3). Several putative leptin binding proteins were detected by leptin-affinity chromatography of which either 80- or 100-kD proteins could be the soluble leptin receptor as approximately 10% of the bound 125I-leptin was immunoprecipitable with leptin receptor antibodies. Significantly higher (P < 0.001) proportions of total leptin circulate in the bound form in lean (46.5 +/- 6.6%) compared with obese (21.4 +/- 3.4%) subjects. In lean subjects with 21% or less body fat, 60-98% of the total leptin was in the bound form. Short-term fasting significantly decreased basal leptin levels in three lean (P < 0.0005) and three obese (P < 0.005) subjects while refeeding restored it to basal levels. The effects of fasting on free leptin levels were more pronounced in lean subjects (basal vs. 24-h fasting: 19.6 +/- 1.9 vs. 1.3 +/- 0.4 ng/ml) compared with those in obese subjects (28.3 +/- 9.8 vs. 14.7 +/- 5.3). No significant (P > 0.05) decrease was observed in bound leptin in either group. These studies suggest that in obese individuals the majority of leptin circulates in free form, presumably bioactive protein, and thus obese subjects are resistant to free leptin. In lean subjects with relatively low adipose tissue, the majority of circulating leptin is in the bound form and thus may not be available to brain receptors for its inhibitory effects on food intake both under normal and food deprivation states.

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.

Effects of prolonged hyperinsulinemia on serum leptin in normal human subjects.

We have studied the effect of prolonged hyperinsulinemia and hyperglycemia on serum leptin levels in young nonobese males during 72-h euglycemic-hyperinsulinemic and hyperglycemic ( approximately 8.5 and 12.6 mM) clamps. Hyperinsulinemia increased serum leptin concentrations (by RIA) dose-dependently. An increase in serum insulin concentration of > 200 pM for > 24 h was needed to significantly increase serum leptin. An increase of approximately 800 pM increased serum leptin by approximately 70% over 72 h. Changes in plasma glucose concentrations (from approximately 5.0 to approximately 12.6 mM) or changes in plasma FFA concentrations (from < 100 to > 1,000 microM) had no effect on serum leptin. Serum leptin concentrations changed with circadian rhythmicity. The cycle length was approximately 24 h, and the cycle amplitude (peak to trough) was approximately 50%. The circadian leptin cycles and the circadian cycles of total body insulin sensitivity (i.e., GIR, the glucose infusion rates needed to maintain euglycemia during hyperinsulinemic clamping) changed in a mirror image fashion. Moreover, GIR decreased between Days 2 and 3 (from 11.4+/-0.2 to 9. 8+/-0.2 mg/kg min, P< 0.05) when mean 24-h leptin levels reached a peak. In summary, we found (a) that 72 h of hyperinsulinemia increased serum leptin levels dose-dependently; (b) that hyperglycemia or high plasma FFA levels did not affect leptin release; (c) that leptin was released with circadian rhythmicity, and (d) that 24-h leptin cycles correlated inversely with 24-h cycles of insulin sensitivity. We speculate that the close positive correlation between body fat and leptin is mediated, at least in part, by insulin.

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.

Dexamethasone stimulates leptin release from human adipocytes: Unexpected inhibition by insulin

In the present study we have examined the effect of dexamethasone on ob gene mRNA expression and leptin release from isolated human subcutaneous adipocytes. Dexamethasone stimulated leptin release from cultured adipocytes in a time‐ and dose‐dependent manner. A two‐fold increase in leptin release was detectable by 36 h of treatment with 10−7 M dexamethasone. Leptin release was preceded by a significant 83±30% increase in ob mRNA after 24 h exposure to the compound. Co‐incubation of cells with dexamethasone (107 M) and insulin (10−7 or 10−9 M) completely blocked the dexamethasone‐stimulated increase in ob mRNA and leptin release. These data demonstrate that insulin and glucocorticoids regulate leptin synthesis and release from human adipocytes in vitro. J. Cell. Biochem. 65:254–258.

Insulin-Like Growth Factor-1 Therapy in Diabetes: Physiologic Basis, Clinical Benefits, and Risks

Salmon and Doughaday [1] proposed in 1957 that the growth-promoting effects of growth hormone in vivo are not direct but are mediated by growth hormone-dependent factors in serum [1]. Insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-2 (IGF-2), collectively known as somatomedins, are the two distinct peptides responsible for the growth-promoting effects of growth hormone [2]. Insulin-like growth factor-1 [3] and somatomedin C [4] are different names for a 70 amino acid, straight chain, basic peptide that is homologous to human proinsulin. The other somatomedin, IGF-2, is a 67 amino acid neutral peptide that is homologous to IGF-1 [2]. Somatomedins were isolated based on three properties: 1) growth hormone-like activities in cartilage (sulfation factor and thymidine factor [2]); 2) mitogenic properties in cell culture systems (multiplication-stimulating activity [5, 6]); and 3) insulin-like activity in adipose tissue and muscle (nonsuppressible insulin-like activity or insulin activity remaining in serum after removal of insulin by insulin antibodies [7, 8]). Human IGF-1 can now be produced in practically unlimited amounts using recombinant DNA technology, and the number of studies of its possible clinical applications is rapidly growing. For example, diabetic patients with extreme insulin resistance had substantial improvement in metabolic control during administration of IGF-1 [9, 10]. More importantly, IGF-1 seemed to improve metabolic control in type II diabetic patients [11]. We describe three main issues. First, we analyze whether using IGF-1 as a substitute for insulin is appropriate, because major differences exist in the kinetic mechanisms of insulin and IGF-1. Second, we review data on the metabolic effects of IGF-1 in normal and diabetic animals. Finally, we describe the metabolic effects of IGF-1 in humans and discuss the potential clinical benefits and risks of IGF-1 therapy in patients with diabetes. Evolutionary Origin of Insulin-Like Growth Factor-1 The concept that IGF-1 mimics insulin action can be defended on the basis of IGF-1 phylogenesis. Chan and colleagues [12] found that insulin and IGF molecules emerged from a common ancestor protein. A gene encoding a polypeptide with a deduced sequence that contains features of both insulin and IGF was detected by DNA cloning in the primitive cephalochordate Branchiostoma californiense [12]. The investigators hypothesized that IGF-1 and IGF-2 genes evolved from the insulin gene by rearrangement of introns (a nucleotide sequence in the DNA of a gene that generally does not code information for protein synthesis and is absent from the mature messenger RNA made from that gene) and subsequent gene duplication, around 300 to 600 million years ago [13]. This intriguing hypothesis explains the known structural similarity between insulin and IGF genes and the homology of these molecules in vertebrates, especially in mammals [14-16]. Kinetic Mechanisms of Insulin-Like Growth Factor-1 The production and targeting to sites of action of insulin and IGFs are different, indicating that the biologic roles assigned to these substances have evolved in different directions [13]. Insulin controls the use of body fuels (amino acids, glucose, and fatty acids). It is produced and stored by specialized, sensing cells in the pancreatic islets that are located in a position that enables secretion directly into the portal system [17]. Thirty to 70% of secreted insulin reaches the microcirculation of the major peripheral organs of insulin action, which are skeletal muscle and adipose tissue [18]. Insulin crosses the vascular barrier with high efficiency, ensuring that most insulin molecules reach specific receptors [19]. The amount of insulin bound is controlled to some extent by receptor affinity that decreases in a curvilinear manner (negative cooperativity) with increased occupancy [20] and by insulin receptor down-regulation [21]. Most circulating IGF-1 is produced by hepatocytes [22, 23] and produced by various other cells to act locally in an autocrine or paracrine manner [24, 25]. The regulation of IGF-1 production is to some extent tissue- or organ-specific. In the liver, production is controlled by pituitary growth hormone [23]. In the extrahepatic tissues, its generation is probably controlled by the concerted action of growth hormone [26] and other pituitary hormones (for example, thyroid-stimulating hormone in the thyroid follicular epithelium and follicle-stimulating hormone and luteinizing hormone in the gonads) as well as by locally produced growth factors (for example, platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor) [27]. None of the cellular sources of IGF-1 can store preformed IGF-1 [28]. The various sources of IGF-1 production, the apparent lack of any known forms of intracellular storage, and the existence of both local and endocrine effects suggest that, unlike insulin, IGF-1 is more like a cytokine than a hormone [29]. The other apparent difference between insulin and IGF-1 is that IGF-1 exists in two forms, a free and a bound form with high affinity to and specificity for various soluble insulin-like growth factor binding proteins (IGFBPs). Our knowledge of all of these proteins is incomplete. Four discrete IGFBPs have been identified in human serum, and a fifth has been identified in human cerebrospinal fluid [29-32]. Together they bind most of the IGF-1 in the circulation, leaving less than 10% of the total serum concentration of IGF-1 in the free form [33]. Under normal conditions, IGFBP-3 is the most abundant binding protein in adult human serum [34, 35] but not in human lymph [36]. After complexing with IGF-1, this protein binds an additional 85-kd acid-labile subunit, and the 150-kd complex circulates in the serum with a half-life of 12 to 15 hours [37, 38]. The 150-kd complex is a major storage form of Ireleased after the complex has been broken down by specific proteases [39]. The production of IGFBP-3 is increased in response to increases in growth hormone [40], insulin [41], IGF-1 [42], and a protein-rich diet [42]. Apart from its functions as an IGF-1 storage and cargo protein, IGFBP-3 can bind to cells and modulate IGF-1-stimulated cell growth [43, 44] and metabolism [44] in vitro. The molecular structure of IGFBP-1 and the sequence of its encoding gene have been established [45, 46]. The levels of this protein in serum appear to be inversely related to prevailing insulin levels [47-50]. Insulin-like growth factor binding protein-1 reaches maximal concentration in the serum during the night when insulin levels are at their nadir [48]; IGFBP-1 apparently serves as a shuttle transporter of IGF-1 from the serum to the interstitial fluid, and it also controls the concentration of free IGF-1 at its site of action [29]. In addition, IGFBP-1 has been shown to modulate the growth-promoting effect of IGF-1 [29]. An increase of IGFBP-1 is associated with growth inhibition of normal tissues [29] and may slow progression of those tumors in which IGF-1 acts as a potent mitogen [51]. The structure of IGFBP-2 and its corresponding gene have also been determined [52]. Its levels appear to be down-regulated by growth hormone [40] and insulin [53] and increased by IGF-1 [54]. The physiologic role of IGFBP-2 is poorly understood, but it may serve as a shuttle transporter of IGF-1 between intravascular and interstitial spaces of target organs. It is the predominant form of IGFBP in cerebrospinal fluid [55]. The regulation of IGFBP-4 is presently unknown, although its molecular structure and the localization of the encoding gene in humans have been reported [56]. Little is known about IGFBP-5. It has been detected in human cerebrospinal fluid and appears to have selective affinity for IGF-2 [57]. Kinetic data for IGF-1 indicate that, despite the common evolutionary origin and the molecular similarity of insulin and IGF-1, their different physiologic assignments result in the development of different systems of generation and delivery to their target organs. Currently used methods of treatment with exogenous IGF-1 rely on doses high enough to substantially increase the level of free IGF-1, but this approach is not physiologic and sometimes produces serious side effects. For example, IGF-1 use in the reversal of catabolic states is substantially compromised by its hypoglycemic effects [58]. In contrast, stimulation of growth (anabolism) may be a side effect when IGF-1 is used to correct hyperglycemia refractory to insulin. The manipulation of IGFBPs may hold the key to future therapy with IGF-1. At present, little is known about how specific sets of IGFBPs modify IGF-1s spectrum of biologic activity. Despite information derived from in vitro experiments, the nature of these effects in vivo is still largely untested. Clinical states associated with high susceptibility to the hypoglycemic effect of IGF-1 provide some information. For example, both growth hormone deficiency and Laron-type dwarfism are characterized by increased susceptibility of affected persons to the hypoglycemic effect of insulin and IGF-1 [59], a phenomenon usually explained by an absent counter-regulatory effect of growth hormone. These two disorders are also characterized by similar alterations in IGFBPs. The concentrations of both the 150-kd complex and its constituent IGFBP-3 are decreased, although the levels of IGFBP-2 are markedly increased [40, 60, 61]. These changes are especially apparent in Laron dwarfs. Interestingly, increases in IGFBP-2 are observed in patients with extrapancreatic (non-insulin mediated) tumor hypoglycemia [54]. The hypothetical role of specific types of IGFBP in regulating the mode of IGF-1 biologic activity (for example, growth-promoting effect versus hypoglycemic action) is presented in Figure 1. Figure 1. Kinetics of insulin-like growth factor-1 in vivo. 1. 2. 3. 4. 5. 6. In Vitro Studies Insulin-like gro

Insulin resistance: site of the primary defect or how the current and the emerging therapies work.

Insulin resistance is one of the cardinal pathophysiological components of the metabolic syndrome, type 2 diabetes, and frequently co-exists with essential hypertension. Although insulin resistance is defined as inadequate target organ (muscle, liver and fat) responsiveness and/or sensitivity to insulin, the primary defect may be located in the target organs themselves or at their remote controller--the central nervous system. One of the ways of resolving this dilemma is studying the mechanisms of action of drugs that have insulin-sensitizing properties. In this brief review we discuss how the known and potential insulin sensitizers: metformin, appetite suppressants, thiazolidinediones, and the new class of centrally acting antihypertensive drugs, I1-receptor agonists, may work.

INSULIN-LIKE GROWTH FACTOR I THERAPY FOR DIABETES MELLITUS ?

I Insulin-like growth factor I (IGF-I) is now produced by recombinant DNA technology, and the number of studies of its possible clinical applications is growing rapidly. Among these studies are trials using IGF-I in cases where insulin action is severely compromised, and the results presented so far are promising. For example, previously published case reports provide descriptions of diabetic patients with extreme insulin resistance in whom significant improvement in metabolic control was achieved during administration of IGF-I (1,2). More importantly, IGF-I has been shown to improve metabolic control in type II diabetic patients (3). These intriguing results raise the question of which differences between insulin and IGF-I action make the use of exogenous IGF-I the better choice for diabetes therapy, and which do not. These important issues are the major focus of this commentary.