Leptin: Lipid metabolism, and clinical applications
Leptin is a hormone secreted by adipose tissue. The discovery of leptin has revolutionized the understanding of obesity and weight regulation providing the first evidence of adipose tissue as an active endocrine organ. The pathophysiology of obesity has entered the "endocrine era" (Flier, 1997). Leptin levels are directly proportional to the amount of body fat in humans. Leptin increases energy expenditure and selectively promotes fat metabolism (Hwa et al., 1997). This hormone takes part in control of body weight by regulating food intake and energy expenditure by a variety of mechanisms, including glucose and fat metabolism modulation (Blüher and Mantzoros, 2009).
Leptin's mode of action has been extensively studied and many aspects still remain unclear. However, it has proven to modulate neuroendocrine, reproductive, angiogenic, and immune function, along with maintaining energy homeostasis. The discovery of this adipokine has provided great advances in the understanding of energy balance and metabolic homeostasis.
Experimentation with exogenous leptin therapy has proved ineffective in directly triggering weight loss in humans. It is proposed that obese individuals develop leptin desensitization, like type II diabetes patients' resistance to insulin, therefore the direct metabolic impact of exogenous leptin may not stimulate weight loss as anticipated. Due to the highly regulated, complex nature of action, the effects of leptin are widespread. The actions on the central nervous system, via the hypothalamus, produce such an integrated response to leptin that its effects may not be pinpointed to one system. However, by elucidating the mechanism of leptin action, perhaps an undiscovered enzyme or part of the process may provide applications of leptin clinically to alter fat metabolism. Clinical uses of leptin are currently effective for leptin-deficient states.
Since leptin discovery, scientists have worked to illuminate the mechanism of leptin action, and have found vast connections with known homeostatic mechanisms. The area for research in leptin has grown monumental, not only for body weight homeostasis, but also for reproductive, immunological, gastrointestinal and neurological functioning. The leptin signal transduction pathway reveals the amount of energy storage available, regulates in a feedback loop fashion, and maintains energy homeostasis.
II. Discovery and Biochemical Properties
Leptin is a 167-amino acid protein whose structure resembles members of the cytokine family. Released by adipocytes, this adipokine revolutionized the understanding of energy homeostasis, showing that fat is an active endocrine organ, not simply a site of energy storage. Leptin is found principally in white adipose tissue (Blüher and Manzoros, 2004). Leptin is found in many other tissues including placenta, mammary glands, testes, ovary, skeletal muscle, pancreas, stomach, heart, hypothalamus, pituitary and many more (Blüher and Mantzoros, 2009). The amount of circulating leptin is directly proportional to the amount of body fat in an individual.
Leptin structure is comprised of 4 alpha helices, with an up-up-down-down topology (Peelman et al., 2004). Leptin has highest structure similarity with cytokines of the IL-6 family, and granulocyte colony-stimulating factor (G-CSF) (Peelman et al., 2004).
Peelman et al. studied the binding sites of leptin in order to develop an antagonist (2004). CRH (cytokine receptor homology) domains were hypothesized as the most important aspect of leptin binding to the long form receptor. The sequence and binding sites of leptin were mapped in reference to known, similar sequences of IL-6 and G-CSFR. The leptin crystal structure was placed over long-chain cytokine structures and binding sites I-III were elucidated.
Binding sites I-III have been observed in other members of the IL-6 family (Peelman et al., 2004). Binding site I on helix D interacts with CRH of IL-6Ra chain (interleukin-6 receptor alpha chain). Binding site II consists of helices A and C and interacts with CRH of gp130. Gp130 is a transmembrane protein found in cytokine receptors that regulates signal transduction. Mutations in binding site II interferes with CRH2 binding, but has limited effect on leptin signaling (Peelman et al., 2004). Binding site III contains the N-terminus of helix D and the loop connecting helices A and B, as well as C and D (Peelman et al., 2004). Site III associates with immunoglobulin-like domain of gp130. A mutation at the N-terminus of helix D produced a successful antagonist. The S120A/T121A mutation in binding site III of leptin completely inhibited signaling capacity without altering binding to CRH2 domain (Peelman et al., 2004). Overall, this antagonist may be used clinically to counteract undesirable actions of leptin, or to provide a tool to define the role of leptin in disease.
Derived from the greek word, leptós, meaning thin, leptin was hypothesized provide the cure for obesity (Halaas et al., 1995). The OB gene-obese gene-found in mice was a model for obesity since the 1970s. A mutation in the OB gene, or autosomal recessive ob/ob mice, produce obese mice with other characteristics. Phenotypically, ob/ob mutant mice are obese, insulin resistant, and infertile (Bowels and Kopelman, 2001; Bray and York, 1979).
In 1979, Bray and York composed a review of genetic models of obesity in experimental animals. The ob/ob genotype-or obese mouse-was an object of interest for studies on genetic transmission of obesity. As an autosomal recessive mutation, the ob/ob genotype has either a deletion or replacement of a nucleic acid on chromosome 6, resulting in an abnormal mRNA, and translation of a defective protein. Characteristics of different experimental animal models of obesity.
Important aspects to note are marked obesity, hyperphagia-increased appetite, hyperglycemia-high blood glucose, insulin resistance, hypothermia, and hypercellular adipose tissue and impaired fertility. The other notable aspect of the striking similarity between the ob/ob obese mouse and db/db diabetic mouse. Therefore, the ob gene product must play a similar role to insulin in regulating food intake, glucose and lipid serum levels, and overall energy homeostasis. Later, the link between diabetes and leptin resistance would be uncovered. The fa/fa recessive mouse also contains similar characteristics aside from hyperglycemia. The fa/fa mouse mutation would prove to be of similar origin as the db/db mouse, with a mutation in the leptin receptor (Bray and York, 1979).
In 1995, an experiment showed how a mutation in the ob gene in C57BL/6J mice (ob/ob mice) causes an obese phenotype, and tested whether administration of the OB gene product-leptin-to mutant and normal mice affected body weight and metabolism (Pelleymounter et al., 1995). Pelleymounter et al. showed how administration of exogenous leptin caused a dose and time-dependent reduction in body weight, in both normal and ob/ob mutant mice (1995). With intraperitoneal injections for 28 days period, the highest concentration of ob protein (10 mg/kg per day) caused the greatest decrease in body weight in ob mice-- 22.2% lowered body weight, and a 52% decrease in food intake. Conversely, mice receiving the lowest concentration of ob protein (0.1 mg/kg per day) showed an increase in body weight by 7.2%. Control mice received PBS (phosphate buffered saline) injections of equal volume, 10 ml/kg, and had a weight increase by 17.13% (Pelleymounter et al., 1995).
Effects of OB protein administration on body weight in A. ob/ob mice B. +/? Mice C. +/+ or wild-type, control mice. Numbers indicate doses of leptin: 0.1, 1.0, 10.0 mg/kg per day. Body weight is represented as a percent difference from day 6 of baseline measurement. Day 1 was the first day of injection. D, E and F represent food intake as percentage of baseline for each group control injected with PBS. D. ob/ob food intake baseline, 4.23 g. E. +/? baseline food intake, 2.97 g. F. +/+ baseline food intake, 4 g. (Pelleymounter et al., 1995).
These breakthrough experiments gave scientists hope that the cure to obesity lay in the ob gene product, leptin. Derived from the greek word, leptós, meaning thin, leptin was hypothesized provide the cure for obesity (Halaas and Friedman, 1995). Halaas et al. experimented with the effects of mouse OB protein and human recombinant OB protein introperitoneal injection on wild-type, ob/ob, and db/db mice (1995). Pair feeding technique showed that ob/ob mice receiving leptin lost more weight than wild type mice given the same food volume (Halaas et al., 1995). Both forms of leptin caused a decrease in body weight and food consumption in wild-type and ob/ob mice. However, db/db mice did not respond to leptin injections, as food consumption and body weight remained stable.
Biological effects of OB protein on food intake and body mass in ob/ob and db/db mice. A and B display ob/ob mice data. C and D display db/db mice. Mice received daily intraperitoneal injections of □= 5µg/g per day OB protein, o = PBS, or Δ= no treatment (Halaas et al., 1995).
This shows the link between insulin and leptin pathways. An insulin resistant, diabetic mouse does not have the ability to respond to exogenous leptin, suggesting that the receptor signal transduction pathways may be related (Campfield et al., 1995). In fact, db/db mice were found to be leptin receptor deficient, denoting the gene for the leptin receptor is coded by the diabetes gene (Chen et al, 1996A).
A study tested the effects of leptin on the central nervous system (CNS) by intracerebroventricular (ICV) cannula injections of OB protein in ob/ob mice (Campfield et al., 1995). Using artificial CSF and saline vehicle injections as controls, the ob/ob mice feeding behavior was compared to OB protein injection over 7 hours. Results showed that OB protein injections into the CNS decrease food intake within 30 minutes. Ob/ob mice receiving leptin stopped eating in the first 30 minutes and did not eat in the remaining 6.5 hours of the experiment. ICV mice receiving vehicle or saline exhibited increased feeding within an hour. Overall, this experiment proves leptin's action on the CNS plays a key role in regulation of food intake (Campfield et al., 1995).
From early studies of leptin, many factors proposed to contribute to this hormone's mechanism of action—central nervous system, feedback regulation, fat store sensing, energy metabolism, and reproduction. This literature review will focus on leptin's mechanism of action, role in lipid metabolism and energy homeostasis, and the clinical applications of leptin affecting energy metabolism.
The Leptin Receptor
In mice, the leptin receptor, Ob-R is coded by the db gene (Chen et al, 1996A). The connection between the ob/ob and db/db genotype was recognized early on, both producing obese genotypes. In 1996, Chen et al. found evidence that the gene for the leptin receptor was encoded in the diabetes gene. This was found using db/db mice and comparing with human cDNA.
The Ob-R protein is alternatively spliced into five known receptors, Ob-R(a-e). The leptin receptor was first isolated in the choroid plexus by gene expression cloning (Tartaglia, 1997). A cDNA library was constructed of the mouse leptin receptor. It was found that mutations in the db gene, yielding an obese, insulin-resistant phenotype, have a defect in the leptin receptor. The db/db mutation, in some cases, has a premature stop codon in the leptin receptor (Chen et al., 1996A). This premature stop codon changes the Ob-Rb receptor to the Ob-Ra receptor, eliminating signal transduction capability of leptin (Friedman and Halaas, 1998).
To analyze the different types of Ob-R gene products, the following method was used to isolate and identify the leptin receptor using the affinity purification method (Li et al., 1998). CNBr activated Sepharose 4B was added to recombinant mouse leptin, and stored at 4º C in PBS sodium azide. One mg of leptin coupled with one ml of Sepharose. Mouse plasma was added to EDTA and centrifuged for affinity purification of the leptin receptor (Li et al., 1998). The prepared leptin was added to mouse plasma and incubated overnight at 4°C. The Ob-R receptor was tagged with two antibodies—antibody A (coding for Ob-R AA sequence 145-158) and antibody B (coding for AA sequence 465-484)—and loaded onto an SDS gel (Li et al., 1998).
The Ob-Rb receptor, or Ob-RL, has a long cytoplasmic region, tyrosine kinase activity, with signal transduction motifs including JAK/STAT box (Friedman and Halaas, 1998). The Ob-Rb is the only isoform with full signal transduction capability (Morris and Rui, 2009). In db/db mice, a premature stop codon is inserted at the 3'- end of the Ob-Rb protein, resulting in the Ob-Ra isoform of the receptor and an obese phenotype (Friedman and Halaas, 1998). The Ob-Rb receptor is found at high levels in the hypothalamus, and proves to be essential for leptin's action regulating energy homeostasis.
All receptor types have identical extracellular sequences, ligand binding sites and differ at the C-terminus. Receptor types Ob-R(a-d) have a single transmembrane domain, and the Ob-Re isoform has none, suggesting it as the soluble form of the receptor. How mutations in db/db and fa/fa mice affect the leptin receptor. The C54B1/Ks db/db mice, there is a premature stop codon in the Ob-Rb form at the 3' end, resulting in the Ob-Ra form of the receptor instead of Ob-Rb (Friedman and Halaas, 1998). Fa/fa mice also have a dysfunctional leptin receptor, due to a premature stop codon. Zucker fa/fa rats have one amino acid substitution of a glutamine for a proline causes a dysfunctional receptor, because proline changes the secondary and tertiary structure of the protein (Friedman and Halaas, 1998).
The Ob-Ra receptor lacks a cytoplasmic domain. It has been found at high levels in the kidneys, lung, choroid plexus, and cerebral capillaries (Ziylan et al., 2009). The considerable concentration of Ob-Ra in the choroid plexus and cerebral capillaries has lead scientists to suspect it may be involved in CNS transport of leptin. Animal models lacking Ob-Ra receptor have decreased transport of leptin as well (Tu et al., 2007).
In 2006, it was found that Ob-Ra receptor regulates clatherin-mediated endocytosis by ubiquitylation (Belouzard and Rouillé, ). Expressing Ob-Ra and Ob-Rb in HeLa cells, both receptor types were ubiquitylated and internalized. Proteasome inhibitors caused inhibition of Ob-Ra internalization, but not Ob-Rb (Belouzard and Rouillé, 2006). When Ob-Ra was constitutively mono-ubiquitylated, proteasome inhibitors no longer prevented endocytosis, which suggests that mono-ubiquination of Ob-Ra regulates the internalization of leptin via clatherin-mediated endocytosis (Belouzard and Rouillé, 2006).
The Ob-Rc and Ob-Rf receptors are located in the cerebral capillaries, however almost nothing is known about the function of these isoforms (Ziylan et al., 2009). The alternate splicing of the Ob-R receptor becomes a source of mutation, and different difficulties in leptin functioning. Clinically, an understanding of the leptin receptor could provide means to alter its expression or affinity binding of leptin and facilitate up regulation or down-regulation of the leptin response, to adjust fat metabolism. Most importantly, the connection between diabetes, insulin and leptin may be found through modifications of receptor gene expression.
Soluble Leptin Receptor: Ob-Re
Leptin circulates in the body in either the free 16kDa form, attached to binding proteins, or bound to a receptor (Li et al., 1998). The binding proteins have been hypothesized as the soluble form of the leptin receptor—Ob-Re.
Since OB-Re is the only isoform lacking a transmembrane domain, it was hypothesized that the OB-Re form of the receptor is the secreted, unbound form (Li, Ioffe, Fidahusein, Connolly, Friedman, 1998). The hypothesis was confirmed using Ob-Re specific antibodies, adding leptin to wild-type and mutant mouse plasma, and observing sucrose sedimentation on a 5-20% gradient. Higher sedimentation occurred in mouse plasma, indicating a higher molecular weight for soluble receptors bound to the ligand. The size of the Ob-Re protein was larger than anticipated by the amino acid sequence, suggesting glycosylation or other post translational modification (Li et al., 1998). To confirm whether Ob-Re is glycosylated, the Ob-Re protein was treated with PNGase F, an amidase that cleaves innermost GlcNAc and asparagine residues. Treatment with PNGase F did cleave Ob-Re into a truncated form, confirming that it is glycosylated.
The purpose of the soluble form of the leptin receptor is still controversial. Many circulating receptors bind their ligand in order to decrease plasma concentration of the ligand, chelating and inhibiting it (Li et al., 1998). Other suggested roles could be to aid in transport across the blood brain barrier, into the CSF, or to foster reuptake after filtration by the kidney. In 2007, it was found that the soluble leptin receptor inhibits transport across the blood brain barrier. Ob-Re has been found to interact with Ob-Ra receptor to inhibit transport across the BBB (Tu et al., 2007).
Leptin Synthesis and Secretion
Leptin is synthesized in white adipose tissue, and to a lesser degree, in brown adipose tissue. Regulation of synthesis occurs at the transcriptional, translational, and post-translational levels. Integration of hormonal signals, and availability of fuel regulate mRNA levels (Lee and Fried, 2009). Insulin increases rate of leptin biosynthesis without altering mRNA levels. Glucocorticoids have been shown to upregulate leptin by enhancing transcription (Lee et al., 2007). Chronic treatment with synthetic glucocorticoid, dexamethasone, causes an increase in mRNA levels. In as little as 2 hours, dexamethasone increases leptin synthesis and release (Lee et al., 2007). Leptin synthesis and secretion are regulated by integration of signals; however, fat cell size has a direct positive correlation with leptin levels.
Overall, women had higher leptin concentrations than males due to the higher percentage body fat women have for childbirth. The difference between male and female leptin release decreases. The larger the fat cell, the more leptin synthesized and secreted.
Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates functions such as transcription, cell motility, cell growth, and protein synthesis (Maya-montiero and Bozza, 2008). mTOR regulates leptin synthesis in adipocytes. "Leptin synthesis in the mature adipocyte is dependent on mTOR. Induction of adipogenesis, which leads to PPARa activation and consequent leptin synthesis, is dependent on PI3K/AKT and mTOR activation" (Maya-montiero and Bozza, 2008).
Neuronal Signal Transduction Pathways of Leptin
Understanding the signal transduction mechanisms is essential in order to comprehend the molecular basis of leptin's physiological action, and to develop clinical advancements to alter energy and fat metabolism. Inhibiting or enhancing particular signal transduction action in the brain or periphery may prove to be of pharmacological significance in individuals with energy imbalances as well as regulating body weight in normal and overweight individuals. While not all pathways have been fully elucidated, far more research has been done on central signaling than action in peripheral tissues. Over the past 15 years, the molecular signal transduction pathway has been studied in detail, and leptin's pathway is closely related to cytokine intracellular signaling. The long leptin receptor, Ob-Rb, is the only form of the receptor that initiates intracellular signal transduction. This receptor is concentrated in the hypothalamus.
Model for neuronal leptin receptor signaling (ObRb). Activation of Ob-Rb by leptin binding causes an increase in JAK2 activity. JAK2 phosphorylates Y985 and Y1138 on the Ob-Rb receptor, leading to phosphorylation of IRS, SHP2, ERK1/2 and STAT3 as well. PI3K, ERK1/2, and STAT3 play a role in gene transcription and other neuronal activity. Negative signaling occurs through socs-3, suppressor of cytokine signaling, and PTP1B (Bjorbaek and Kahn, 2004).
Like cytokine signaling, leptin induces the JAK/STAT pathway. When leptin binds to the Ob-Rb homodimer, JAK2 (Janus activating kinase 2) is activated, which leads to Ob-Rb receptor tyrosine auto phosphorylation (Friedman and Halaas, 1998). JAK2 stimulates tyrosine phosphorylation at multiple sites on the receptor. Tyrosine amino acid 985 and 1138 have src homology 2 (SH2 domain), a binding motif for proteins containing SH2 domain (Bjorbaek and Kahn, 2004). JAK2 stimulates a variety of intracellular signaling pathways in neuronal cells by way of STAT3, STAT5, SH2-containing tyrosine phosphatase 2 (SHP-2), insulin receptor (IRS), and protein tyrosine phosphatase 1b (PTP1B).
STAT3 becomes activated with phosphorylation of tyrosine 1138 on Ob-Rb receptor. It dissociates and forms a dimer in the cytoplasm where it translocates into the nucleus to regulate gene expression (Bates et al., 2003). Scientific evidence has proven that the STAT3 pathway has the greatest impact on energy homeostasis (Bates et al., 2003). In vitro, leptin binding to receptor activates Stat 1, 2 and 3, but only Stat 3 is activated in vivo (Friedman and Halaas, 1998); "however, leptin primarily stimulates STAT3 and STAT5 phosphorylation in the hypothalamus in animals" (Morris and Rui, 2009 pgE1249). Disruption of the STAT3 binding site, or inhibition of neuronal STAT3 results in severe obesity and hyperphagia (Morris and Rui, 2009; Bates et al., 2003).
In vitro, leptin activation of STAT3 activates the proopriomelanocortin (POMC) promoter in cultured cells (Morris and Rui, 2009), as well as agouti-related protein (AgRp). The endocrine actions of POMC plays a will be discussed in a latter section. POMC neurons in the Arculate Nucleus (ARC) of the hypothalamus are anorexigenic—or induce anorexia. POMC neurons release POMC as well as cocaine- and amphetamine-regulated transcript (CART) (Morris and Rui, 2009). AgRP/NPY neurons co-express neuropeptide Y and Agrp, both orexigenic neuropeptides that increase food intake. Leptin activates POMC neurons and inhibits AgRP/NPY neurons, to suppress food intake.
SHP-2, SH2-containing protein tyrosine phosphatase 2, binds tyrosine 985 on Ob-Rb and activates the MAP kinase pathway (MAPK). ERK becomes phorsphorylated and activates several transcription factors that enters the nucleus to regulate gene transcription (Bj?rbaek and Kahn, 2004). Experiments with SHP-2 deletion mutants result in leptin resistance and obesity, suggesting SHP2 mediates anorexigenic action of leptin (Morris and Rui, 2009).
Leptin also stimulates insulin receptor substrate (IRS), which stimulates the phosphoinositide 3-kinase (PI3K) pathway. Inhibition of PI3K in the brain eliminates leptin's action to decrease food intake and weight gain (Morris and Rui, 2008), as well as leptin's action to suppress lipogenesis in white adipose tissue (Bai et al., 2008). Leptin's direct action on the insulin signaling pathway provides evidence for cross talk between the two hormones.
SH2B1 is an SH2 and plecstin homology domain-containing adaptor protein that positively regulates leptin action. SH2B1 works through two mechanisms of signal transduction regulation. First, leptin stimulated JAK2 autophosphorylation of tyrosine 813 allows SH2B1 to bind the phosphorylated site and enhance JAK2 activity. Secondly, SHB2 binds directly to IRS-1 and IRS-2 and subsequent enhancement of PI3K pathway activity (Morris and Rui, 2009).
The signaling pathways described above indicate positive regulation of leptin's anorexigenic activity. However, two other signaling cascades have been found as negative regulators for leptin action: suppressor of cytokine signaling-2 (SOC3) and protein tyrosine phosphatase 1B (PTP1B). SOC3 transcription results from negative feedback of the STAT3 cascade. By binding JAK2, SOC3 directly inhibits JAK2 activity. Deletion of SOC3 causes slight increase in leptin sensitivity, and protects against diet-induced obesity (Morris and Rui, 2009). Levels of hypothalamic SOC3 significantly increase in obese, leptin-resistant animals, suggesting that SOC3 may contribute to leptin resistance (Morris and Rui, 2009). PTP1B negatively regulates leptin signaling by dephosphorylation of JAK2.
A model for leptin signaling. JAK2 dependent pathways include activation of IRS, SH2B1, MAPK, STAT5, and Akt. Leptin activation of Akt (protein kinase B) inhibits TCS 1 and 2 (tuberous sclerosis complex 1 and 2) which activates mTOR (rapamycin), and S6K (ribosomal S6 kinase). The JAK2 independent pathway that works synergistically to regulate energy homeostasis and body weight activates Src, a kinase pathway regulating STAT3 activity (Morris and Rui, 2009).
Recent discoveries find both JAK2 dependent and independent pathways for regulation of energy homeostasis. JAK independent pathways include activation of CaMKK2/AMP-activated protein kinase pathway (AMPK), which inhibits acetyl-CoA carboxylase (ACC). AMPK is a kinase that senses the energy state of the cell, as it is activated by increased AMP concentrations, or a low energy state. ACC is the rate limiting enzyme of fatty acid synthesis. Inhibition of ACC provides evidence for decreased lipid synthesis. Another JAK2 independent pathway is activation of Src, which enhances STAT3 activity (Morris and Rui, 2009).
An important JAK2 dependent pathway that enhances weight loss is the mTOR pathway. PI3K activates Akt—a serine-threonine specific kinase, or protein kinase B—which inhibits tuberous sclerosis complex 1 and 2 (TCS1/2) (Maya-Monteiro and Bozza, 2008). TCS1 and 2 inhibit mTOR, therefore leptin inhibition of TCS 1/ 2 activates mTOR (Maya-Monteiro and Bozza, 2008). mTor works with Raptor protein, and GBL to activate S6K, to enhance weight loss. Peripherally, activation of the mTOR pathway via leptin enhances immune function. The mTOR pathway exhibits a function of leptin that has pleotropic effects depending on the tissue it acts upon.
Peripheral Leptin Signaling
While studies show central leptin signaling to play a major role in regulation of food intake and energy metabolism, peripheral actions of leptin may be equally significant in controlling energy homeostasis (Guo et al., 2007). Due to the fact that the ObRb receptor has the highest concentrations in the brain, and has direct signal transduction capability, the central mechanism of leptin has been studied more extensively, leaving peripheral actions of leptin unclear.
In peripheral tissue, the leptin receptor isoform with the highest concentration is OB-Ra, or the short isoform. Ob-Ra mRNA has the highest concentration in the lungs, kidney, and lymph nodes, and lower mRNA levels in the heart, liver, spleen, white adipose tissue (WAT), adrenals, testes, and skeletal muscle (Bjorbaek and Kahn, 2004). Ob-Rb mRNA can be detected in the lungs, kidneys, adrenals and lymph nodes. Presence of Ob-Rb mRNA at lower levels occurs in the liver, brown adipose tissue, WAT, and skeletal muscle (Bjorbaek and Kahn, 2004). While mRNA can be detected in these areas, detection of the receptor proteins is very difficult due to the low expression level peripherally. However, the variety of tissues with leptin receptor mRNA suggests receptors are present and illustrates the diverse actions of leptin affecting all body systems. One could postulate that receptors in the lungs may mediate the increased O2 expenditure, receptors in the lymph nodes may mediate immune function, and adrenals to facilitate SNS outflow.
When a chemical sympathectomy is preformed on rats, the effect of SNS outflow can be measured under changing conditions. After prolonged ICV administration of leptin in chemically sympathectomized rats, food intake and weight loss were measured to see importance of SNS activation on leptin regulation of energy (Dobbins et al., 2003). Cutting off SNS outflow in rats resulted in less weight loss compared to controls, however food intake remained identical. This experiment demonstrates the importance of increased SNS outflow in leptin's metabolic activity.
Guo et al. studied the role of peripheral signaling in leptin's action regulating whole body energy metabolism in vivo (2007). Scientists created a mouse model, Cre-Tam, with in tact central leptin receptors, and deleted peripheral signaling in adipose tissue, the liver and small intestine. Deletion of peripheral signaling was done using a taximofen (TAM)-inducible Cre-LoxP system (Guo et al., 2007). These mice were hyperleptinemic, or had elevated levels of leptin, and a 2.3-fold increase in post-translational production of leptin. While there was elevated levels of leptin, the level of free leptin remained the same, due a marked increase of plasma bound leptin. However, there was no change in energy balance, insulin sensitivity, or temperature regulation. The results of this study concluded that disturbance of peripheral signaling does not affect overall energy homeostasis, and that disrupting the short-loop negative feedback in adipocytes causes unregulated leptin production, and hyperleptinemia (Guo et al., 2007). While results of this study show a lack of importance of peripheral leptin action in Cre-Tam mice energy metabolism, peripheral signaling in humans may be strikingly different. Further studies are necessary to confirm leptin's action in peripheral tissue in humans on whole body energy homeostasis.
IV. Tissue Distribution
It was proposed that leptin directly affects the signaling pathways in skeletal muscle (Bjorbaek and Kahn, 2004). A new signal transduction cascade of the leptin receptor was identified in skeletal muscle—AMP-activated protein kinase (AMPK) (Bjorbaek and Kahn, 2004). AMPK is a serine/threonine kinase that is activated by an increased intracellular ratio of AMP:ATP or upstream signals, and regulates substrate metabolism. AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC) and decreases production of its product, malonyl CoA. ACC catalyzes the rate limiting step of triglyceride synthesis. Decreased concentration of malonyl coA disinhibts carnitine palmitoyl transferase-1 (CPT-1). CPT-1 is required for fatty acid transport into the mitochondria, and thus stimulates fatty acid oxidation in mitochondria (Bjorbaek and Kahn, 2004).
Model for two mechanisms of leptin regulation of adenosine monophosphate protein kinase (AMPK) and fatty acid oxidation in skeletal muscle. First, leptin activates Ob-Rb receptor and increases AMP/ATP ratio to activate AMPK. Secondly, leptin increases sympathetic nervous system (SNS) outflow via CNS penetration. a-adrenergic receptors are activated and increase AMPK activity. AMPK phosphorylates acetyl-CoA carboxylase (ACC), and inhibits its formation of malonyl-CoA. Decreased malonyl-CoA disinhibits carnitine palmitoyltransferase 1 (CPT-1), which inhibits transport of fatty acids into mitochondria. (Bjorbaek and Kahn, 2004).
Leptin injection in the hypothalamus, or incubation of muscle with leptin confirms that leptin causes increased fatty acid oxidation in muscle by activating AMPK, and inhibiting ACC (Minokoshi and Kahn, 2003). These regulatory effects require the Ob-Rb receptor. Further studies show that leptin has a great effect on fat metabolism in resting muscle, but not contracting muscle (Lau et al., 2001). The level of palmitate uptake in leptin treated soleus was less than in leptin-untreated muscle, showing that leptin inhibits fatty acid uptake in muscle.
In isolated adipocytes, leptin inhibits lipogenesis and induces lipolysis (Morris and Rui, 2009). Wang, Lee and Unger found that leptin acted to decrease fat stores without increasing circulating free fatty acids (FFA), ketones or other lipids (1999). Details on leptin effects on lipid metabolism are detailed in later sections.
The way leptin chronically affects the vasculature and the heart is currently unknown. However, the enzyme PTP1B has become a new target of interest for treatment of obesity and type II diabetes (Belin de Chantemele, 2009). Leptin has been shown to decrease vascular adrenergic reactivity to mediate leptin increases in mean arterial pressure (MAP). Using PTP1B knockout mice, leptin sensitivity was measured by how subcutaneous leptin injections affected cardiovascular function and body weight. After 9 days of leptin infusion at 10µg/kg per day, there was a significant increase in MAP in knockout mice. However, it was determined that the PTP1B deletion mediates the sympathetic stimulatory effects of leptin, blunting increases in blood pressure and vasoconstriction (Belin de Chantemele, 2009).
Leptin has also been found to decrease plasma atrial natriuretic peptide (ANP), which is an important regulator of blood pressure (Yuan et al., 2010). Released from atrial myocytes, ANP antagonizes the renin-angiotensin system, and causes dieresis, natiuresis and vasodilation. Inhibition of ANP release results in hypertension and cardiac hypertrophy. Yaun et al. found that intravenous injection of rat leptin into rats decreased ANP levels and elevated blood pressure (2010). However, leptin induced elevation of blood pressure was transient. This effect was blunted by pretreatment with N?-nitro-L-arginine methyl ester, an inhibitor of nitric oxide. Therefore, leptin reduces ANP in plasma by a nitric oxide dependent mechanism (Yuan et al., 2010). Since obese individuals tend to have increased blood pressure and elevated leptin levels, ANP may be the mediator of leptin-induced hypertension.
Bray and York (1979) observed hypertrophy and hyperplasia of pancreatic islet B cells in ob/ob and db/db mice. Lipotoxicity of B cells has been identified as the cause of diabetes in Zucker fa/fa rats as well (Unger et al., 1999). B cells in ob/ob mice are highly vascularized and degranulated, and the high level of serum insulin suggests there is increased synthesis and secretion of insulin (Bray and York, 1979).
Leptin and Central Nervous System
Transport in the Central Nervous System
Feedback loops regulate energy homeostasis by integrating external and internal stimuli through the CNS. With peripheral signals relaying back to the brain, modifying expression of hormones and neurons. Leptin is released from adipocytes and acts on the CNS, principally via the hypothalamus—through a saturable transport system (Bowles and Kopelman, 2001). This peripheral signal relays back to the brain, and must cross the selective blood brain barrier or enter the cerebrospinal fluid (CSF) to access the hypothalamus. In the CNS, leptin activates or inhibits neurons, modifies gene expression and sends efferent signals to alter energy homeostasis. While leptin does act on peripheral tissues where receptors are present, its transport into the CNS is imperative for functional regulation of energy metabolism.
Two major structures mediate regulatory protein transport into the CNS: choroid plexus epithelial cells, and blood brain barrier (BBB) endothelial cells. The highest rate of leptin transport is in the hypothalamus, and the lowest rate is in the cerebral cortex. However, with changing concentrations of plasma leptin, uptake in different parts of the brain changes due to varying saturability of brain tissue. High serum leptin levels cause the highest uptake in the pons and medulla, and lowest uptake in the hypothalamus (Ziylan et al., 2009).
The choroid plexus, named after the latin words for delicate knot, is located in four places in the brain (one per ventricle) and separates the cerebrospinal fluid (CSF) from blood. Transport of leptin into the CSF occurs at a much higher rate--18-50 times faster—than transport across the blood brain barrier. Leptin transport into the CSF occurs at a rate comparable to Na+, Cl- uptake (Ziylan et al., 2009). The receptor that mediates leptin transport into the CSF is unknown, but may be endocytosis. There is a presence of tight junctions in the chroroid plexus, and bulk flow has been hypothesized as a mode for leptin transport as well (Ziylan et al., 2009). Overall, there is clear importance of the choroid plexus in leptin transport into the CNS.
Leptin transport through the BBB occurs in cerebral capillaries, at a rate much slower than through the CSF, a rate comparable to insulin, growth factor and vasopressin. However, transport into the hypothalamus occurs at a much faster rate than anywhere else across the BBB (12-37 times faster) (Ziylan et al., 2009). Leptin also accesses the hypothalamus by diffusion from the CSF.
The mechanism of leptin transport across the blood brain barrier and CSF is uncertain. It has been hypothesized that the short form receptor, Ob-Ra, mediates leptin transport (Friedman and Halaas, 1998). The most recently published studies on leptin transport into the CNS hypothesized reliance on endocytosis, with possible dependence on the Ob-Ra receptor. In 2010, a study testing leptin uptake in cerebral endothelia using Ob-R receptor mutants, and examined uptake in presence of different inhibitors of endocytosis (Tu et al., 2010). The Ob-R mutants had varying cytoplasmic domain lengths, to see if endocytosis depends on intracellular sequences. Results concluded that the cytoplasmic region of the Ob-R receptor is not necessary for clathrin-dependent endocytosis of ObRa (Tu et al., 2010). While both the Ob-Rb and Ob-Ra receptors were endocytosed, this study showed that intracellular JAK/STAT activity is not necessary for internalization of leptin at the BBB.
The hypothalamus has been shown to be the main site regulating energy homeostasis in the CNS, regulating hunger, thirst, body temperature, fatigue, and sexual desire. Overall, when leptin acts on hypothalamic neurons, there is an increase in synapses on neurons secreting anorexigenic neuropeptides, and decreased synaptic activity of neurons secreting orexigenic peptides like neuropeptide Y (NPY) (Kelesidis et al., 2010).
Studies show that the hypothalamus has the largest uptake of leptin in brain tissue (Ziylan et al., 2009). Leptin receptor Ob-Rb mRNA is most concentrated in the hypothalamus at the arculate nucleus (ARC), dorsomedial (DMH), ventromedial (VMH), and ventral premamillary nuclei (PMV) (Bj?rbaek and Kahn, 2004).
Leptin on hypothalamic circuits affecting energy balance. In the ARC, leptin stimulates anorexigenic POMC/CART neurons, and inhibits AgRP/NPY orexigenic neurons. In the PVH/VMH, melanocortin 4 receptors (MC4R) become activated by POMC-released a-melanocortin stimulating hormone (a MSH). In the lateral hypothalamus, neurons release melanin concentrating hormone (MCH), which increases feeding behavior. Orexins released from the lateral hypothalamus act to regulate wakefulness and energy balance (Flier, 2004).
In the ARC, different populations of neurons respond to leptin. Leptin action on POMC neurons has been postulated to be a key regulator of energy homeostasis. Pro-opiomelanocortinin (POMC) neurons produce anorexigenic peptide a-melanocyte-stimulating hormone (a-MSH), which act on 3/4 melanocyte receptors (MC3/4-R). Cocaine and amphetamine-regulated transcript (CART) is another inhibitor of feeding, and neurons expressing CART overlap with ARC neurons (Bjorbaek and Kahn, 2004).
Thornton et al. found that leptin directly regulates expression of POMC mRNA in the hypothalamus of ob/ob mice (1997). Using in situ hybridization of mice hypothalamus, fluorescent labeling of POMC mRNA in both wild-type and ob/ob mice, the level of mRNA was measured by counting fluorescent grains in a photomicrograph. Results show the highest amount of POMC mRNA in the control mice, especially in the retrochiasmatic area (RCh), and in the arcuate nucleus. The low amount of POMC mRNA in ob/ob control mice compared to leptin treated ob/ob mice proves that administration of leptin up regulates POMC mRNA transcription (Thornton et al., 1997). Limits of this study include the method of in situ hybridization, which does not necessarily reflect in vivo action, and the use of mice hypothalamus instead of human tissue. However, future studies would confirm leptin's direct action on POMC neurons.
In the orexigenic Agouti-related peptide neurons (AgRP neurons) of the ARC nucleus, leptin exhibits inhibitory effect (Morris and Rui, 2009). AgRP neurons release orexigenic AgRP and neuropeptide Y (NPY). NPY stimulates feeding, and AgRP protein acts as an antagonist on MC 3 /4 receptors in the VMH/PMH. AgRP neurons also inhibit anorexigenic activity of POMC neurons by releasing GABA, an inhibitory neurotransmitter. Leptin acts to stop inhibitory action on POMC neurons, and decreases AgRP neuronal excitability (Morris and Rui, 2009).
In the VMH/PMH, MC3/4 receptors are activated by leptin-induced secretion of aMSH from POMC (Forbes et al., 2001). aMSH inhibits food intake, and also diffuses to the periphery where it binds MC-Rs on adipocytes to induce fat mobilization (Forbes et al., 2001). Brain derived neutrotropic factor (BDNF), which binds to receptor TrkB, can also be found in the ventromedial nucleus of the hypothalamus, in neurons associated with satiety. BDNF release results from MCR4 signaling and nutrition. Heterozygous BDNF mice mutants are obese with increased appetite (Flier, 2004).
In the lateral hypothalamus, neurons release melanin concentrating hormone (MCH), a hormone that induces sleep and increases feeding behavior. Deletion of MCH gene produces a lean, hypophagic mouse with increased energy expenditure. Orexins released from the lateral hypothalamus act to regulate wakefulness and energy balance (Flier, 2004). Orexins act on OX2R receptors which inspire Na+/Ca+ channel exchange in GABA releasing neurons. GABA in the brain increases feeding behavior (Flier, 2004).
Signaling in the hypothalamus leads to an increase in phosphodiesterase 3B (PDE3B), which causes a decrease in hypothalamic cyclic adenosine monophosphate (cAMP) (Bj?rbaek and Kahn, 2004). Decreased cAMP in the hypothalamus has been linked to hypertension (Schmid et al., 1979). Leptin induced PI3K activity has been shown to mediate sympathetic nervous system outflow (Bjorbaek and Kahn, 2004).
Growth hormone (GH) stimulates body growth and also plays a role in metabolism. Carro et al. inquired about the induction of GH in response to changing energy states, and nutritional status, and whether leptin was involved (1997). Using normal adult rats, leptin was administered in the fed and fasted state, and GH level was measured every 15 minutes for 6 hours (Carro et al., 1997). Results show a profound decrease in GH secretion in the fasted state, and that leptin reversed this inhibitory effect, causing an increase in GH levels. Leptin administration to normal, fed rats, did not alter GH secretion (Carro et al.,1997). This experiment denotes the importance of leptin in the fasted state over the fed state. Leptin causes an increase in GH only results in the epinephrine and leptin have an interesting relationship. Ob/ob mice exhibit decreased sensitivity to epinephrine (Bray and York, 1979). In adipose tissue, cAMP concentrations did not increase to the level of normal mice after application of epinephrine. In vitro, B-agonists and other activators of adenylate cyclase inhibit insulin-stimulated leptin release, but also stimulates lipolysis and release of FFA (Selenscig et al., 2009). Long term activation of B-adrenergic receptors results in a decrease of leptin mRNA (Lee and Fried, 2009).
Epinephrine causes an increase in leptin transport through the BBB. In the periphery, leptin stimulates epinephrine release, and epinephrine inhibits leptin release but also inhibits appetite. In the brain, leptin inhibits epinephrine secretion, and epinephrine stimulates appetite. "Thus, by stimulating leptin transport across the BBB, peripheral epinephrine would facilitate leptin-induced suppression of CNS epinephrine release and so intensify its anorectic action" (Banks, 2006).
Leptin and Insulin
Leptin and insulin are both hormones of energy metabolism—leptin regulating fat, and insulin regulating serum glucose levels. In the discovery of leptin, the two models of obesity, ob/ob and db/db, exhibited nearly identical phenotypes, with early onset of weight gain and insulin resistance (Bray and York, 1979). While the db gene was a proposed mutant model for diabetes, it actually codes for the leptin receptor (Chen et al., 1996A). Db/db mice are leptin receptor deficient, and severe obesity may be a cause of the diabetic phenotype. Hence, ob/ob mice may develop insulin resistance in the same fashion, because both models are leptin- signaling deficient. Consequent studies have measured the interactions between the ob gene product, leptin, and insulin. Interactions between leptin and insulin include, common signal transduction interaction, leptin receptor encoded in the db gene, and new interest in leptin as treatment for diabetes (Hedbacker et al., 2009).
Pelleymounter et al. showed how leptin decreases serum insulin and glucose levels. The ob/ob PBS control exhibits elevated insulin and glucose levels, denoting insulin resistance. However, upon administration of the lowest dose of leptin (0.1 mg/kg), there is a dramatic drop in glucose, and a similar drop in insulin at a higher leptin concentration of 1 mg/kg. Leptin enhances glucose metabolism, and decreases serum insulin (Pelleymounter et al., 1995). With even the smallest amount of leptin, the circulating insulin becomes more effective, and serum glucose levels drop and these results suggest that leptin enhances insulin-sensitivity. Use of leptin to treat type 2 diabetes will be described in the clinical applications section.
Leptin and Energy Homeostasis
Leptin's role in regulation of energy homeostasis and fat metabolism has proved most important in a state of starvation, rather than with energy excess, suggesting leptin evolved as protection from starvation (Unger, 2005). Since leptin concentrations are directly proportionate to fat mass, this hormone serves as an indicator of fat storage. With starvation comes a drop in leptin concentration, sensed in the hypothalamus, wherein feedback loops adjust secretion of other hormones, inducing a state of energy conservation (Bowels and Kopelman, 2001).
On the right, an increase in fat cells signals an increase in leptin, signaling the hypothalamus to inhibit ArT neurons, stimulate MSH, release corticotrophin releasing hormone (CRH), and induce a variety of other weight reducing actions. CRH induces a stress response, and decreases feeding (Flier, 2004). The end result of a high leptin concentration becomes increased energy expenditure, increased sympathetic tone, and decreased food intake (Friedman and Halaas, 1998).
In weight loss, decreased leptin circulates, causing activation of NPY to increase food intake. Increased NPY is a signal for starvation that activates feedback loops to decrease energy expenditure, decreased GH, GHRH and GnRH release, increase CNRH and parasympathetic tone (Friedman and Halaas, 1998).
Leptin: Signal in Starvation
It has been suggested that leptin evolved as an adaptation during a time of starvation to regulate energy expenditure. In starvation, the evolutionarily favorable response prevails, maintaining vital organ function, utilizing as little energy as possible, and thus maintaining energy balance to ensure survival. With depleted fat stores, a decreased leptin level circulates in starvation. Therefore, reverse leptin signaling causes decrease fat breakdown, decreased energy expenditure, and increased food intake. Decreasing fat breakdown, ensures fat stores remain intact. With less energy reserve sensed, a decrease in energy expenditure prolongs life. If there is no food present, a decreased appetite is favorable as well. Evolutionarily, it is favorable to enter an energy conserving state when in starvation (Bowels and Kopelman, 2001).
In a state of starvation, reproductive function is not essential for survival. Therefore, in leptin deficient individuals, reproductive function suffers as exhibited in ob/ob mice (Bray and York, 1979). Maintaining muscle mass is important in starvation, and leptin works to selectively breakdown fat stores before muscle (Ramsay, 2003). Loss of muscle mass becomes dangerous in starvation, as it can deplete cardiac muscle tissue, posing increased risk of mortality by a cardiovascular event.
Leptin treatments have been used effectively to treat anorexic amenorrhea (Chan and Mantzoros, 2005). Since anorexic individuals have depleted fat stores, they have decreased leptin circulating. Thus, leptin's role in regulating the menstrual cycle becomes disrupted in a starvation state and anorexic women cease to have a menstrual period. By clinically administering leptin, amenoreah in anorexic women can be reversed (Chan and Mantzoros, 2005).
VI. Metabolic Effects of Leptin
Mechanisms of Energy Homeostasis
The integrated action of leptin decreases body weight, and maintains fat storage. While most of these actions of leptin occur via the CNS, every cell on the body that has a functional leptin receptor also has a mechanism to increase energy expenditure and regulate fat storage. As described previously, increased sympathetic outflow also enhances leptin action. The major mechanisms of energy homeostasis are as follows—decreasing food intake, increased temperature, increased fatty acid breakdown, decreased fatty acid synthesis, and overall increase in body weight loss.
Leptin and Food Intake
Food intake is one of the most integrated behaviors in humans. Decreased food intake was one of the first functions of leptin recognized in. Pelleymounter et al. found a decrease in food intake by 52.6% in ob/ob mice with administration of leptin for 28 days (1995). Hypothalamic injections of leptin caused a complete cessation of food intake for 7 hours in ob/ob mice (Campfield et al., 1995).
Leptin modulates food intake by hypothalamic signaling. When the hypothalamic orexigenic and anorexigenic neurons are damaged, hyperphagia results, or an inability to regulate food intake. It is proposed that leptin is an afferent satiety signal, telling the brain it has enough energy storage, and need not use available energy to find food. Through inhibiting orexigenic neurons in the hypothalamus, and inducing the anorexigenic, leptin decreases the need for food (Unger, 2004).
Recent research shows a connection with the dopaminergic system and leptin's action inhibiting food intake. DiLeone recognized that leptin receptors in ventral tegmentum (VTA) may mediate leptin's action in feeding behavior (2009). The VTA comprises the mesolimbic and dopaminergic system which links to reward circuitry, cognition, motivation and addictive behaviors. "Lack of control over food intake bears resemblance to drug addiction, where loss of control over behavior leads to compulsive drug use" (DiLeone, 2009). Direct effects of leptin on dopamine neurons have been shown in recent studies. Showing pictures of food to leptin-deficient individuals caused hyperactivity in the VTA, and high ratings given to the pictures. After leptin administration, VTA stimulation showed normal levels of stimulation, and individuals gave lower preference ratings to the pictures (DiLeone, 2009). Further research on leptin's action on dopaminergic neurons may provide a mechanism to treat obesity.
Leptin and Temperature Regulation
Ob/ob mice display hypothermia, or decreased body temperature within the first 10-14 days of life, after brief exposure to cold (Bray and York, 1979). This is due to the lack of "warming" effect of leptin action. Pelleymoutner et al. (1995) confirmed that the lowest dose of leptin to ob/ob mice raised body temperature equal to control lean mice.
Leptin administration on core temperature of +/+, ob/ob, and +/? mice. The ob/ob mouse has a lower control body temperature than the other two genotypes, and temperature increases to normal levels with administration of leptin (Pelleymounter et al., 1995).
In PBS controls Core body temperature of ob/ob mice is significantly lower than wild-type, +/+ mice. Leptin administration on control, +/+, mice showed no change in body temperature. However, administration of leptin to ob/ob mice at the highest dose, 10 mg/kg, caused temperature to elevate to a normal level just under 37º C (Pelleymounter et al., 1995). These results suggest that leptin regulates temperature in normal individuals.
Leptin increases body temperature by increasing the level of uncoupling protein (UCP) in cells, especially brown adipose tissue (BAT). These uncoupling proteins create a channel in mitochondria for protons to flow from the cytosol to the mitochondrial matrix. This proton flow disrupts concentration gradient, decreasing the effective of ATP generation of mitochondria, and releasing the energy in the form of heat. Central infusions of leptin, cause an increase in BAT uncoupling protein UCP-1 and UCP-2 (Zhou et al., 1997).
Environmental temperature has been shown to influence leptin responsiveness in wild-type mice (Harris et al., 2007). Depending on whether mice received a high fat (HF) or low fat (LF) diet, metabolic effects differed in 18ºC, 23ºC, and 27ºC environments. Mice given a LF diet were the only subjects to lose body fat with leptin infusions, and only in the 23ºC, room temperature environment (Harris et al., 2007). HF diet mice in a hot, 27 ºC environment accumulated more fat. HF diet mice were models for diet-induced leptin resistance. Therefore, diet-induced leptin resistance is only partial resistance in a hot environment (Harris et al., 2007).
Leptin and Fuel Selection
Overall, leptin selectively enhances lipid metabolism and release of FFA into plasma. Hwa et al. (1997) found that leptin selectively alters fuel selection: enhancing fat metabolism and decreasing carbohydrate metabolism in ob/ob mice.
Selective increase in free fatty acid and decrease in carbohydrate serum levels in leptin treated ob/ob mice. Serum free fatty acids and glucose serum levels in ob/ob mice 3 and 24 hours after dosing with leptin (white bars), or saline (black bars). (Hwa et al., 1997).
Dose-dependent effects of leptin on percent of energy derived from carbohydrate (black) or fat (white) in ob/ob mice. (Hwa et al., 1997).
In untreated ob/ob mice, 95% energy derived from carbohydrate and 5% from fat. Twenty-three hours after leptin injection of 3 mg/kg, 56% energy was carbohydrate derived, and 44% was fat derived. Three hours after leptin injection, there was a significant increase in FFA circulating, and no change in carbohydrate circulation. Twenty-four hours after injection, leptin's effect on FFA in serum decreased, but a lower carbohydrate circulation was maintained. Due to the short half-life of leptin, the effects after 3 hours are due to leptin binding with receptor. Therefore, the increase in FFA is a direct effect of leptin action. Leptin's effect on carbohydrate metabolism was maintained over 24 hours, long after protein degradation. Long term effects on carbohydrate metabolism must be due to an indirect of leptin. Most likely, the sustained decrease in serum glucose may be due to enhanced insulin- sensitivity by leptin (Hwa et al., 1997)
Leptin and Overall Energy Expenditure
No effect on basal metabolic rate has been seen with leptin (Soares et al, 2000). Johnstone et al. (2005) showed that fat mass and other factors affected basal metabolic rate, but that leptin did not.
Measuring oxygen consumption, respiratory quotients, Hwa et al. used ob/ob mice to study leptin's effects on energy metabolism (Hwa et al, 1997). Leptin treated rats exhibited an increase in oxygen consumption. Increased oxygen consumption may be due to increased sympathetic outflow from CNS in response to leptin, as well as thermogenic effects.
Respiratory quotient (RQ)—a measure of CO2:O2—decreased in leptin treated ob/ob mice from .99 to .87. A RQ of 1.0 represents pure carbohydrate metabolism, and .7 denotes pure fat metabolism (Hwa et al., 1997). The decrease in respiratory quotient denotes change in fuel selection.
Due to the fact that leptin is a hormone, some effects occur almost immediately, and others develop over a long period of time. Like hormones in development of reproductive maturity, gradual increases in a hormone from youth eventually cause effects that may seem sudden, but have gradually accumulated to target concentrations required for sexual maturity. Leptin is a hormone involved in reproduction as well, which indicate that mechanisms of energy regulation and metabolism may develop in a similar long term manner. Essentially, leptin has short term effects to increase fat metabolism, and long term effects to decrease body weight. Acting on a variety of receptors, leptin enhances fat metabolism in the periphery, and switches balance of fuel from carbohydrate to fat.
VII. Leptin and Lipid Metabolism
In studies done on mice and humans, leptin administration shows an increase in FFA and a decrease in body fat (Hwa et al, 1997), which suggests activation of lipolysis, or lipid breakdown. In a study inducing sustained hyperleptinemia in rats, results show disappearance of fat to maintain of body weight (Chen et al., 1996B). By infusing recombinant adenovirus containing rat leptin cDNA, sustained hyperleptinemia of 8 ng/ml was maintained for 28 days. Postmortem examination showed absence of subcutaneous, visceral, retroperioneal, and epididymal fat stores (Chen et al., 1996B). In pair-fed controls given recombinant adenovirus with B-galactosidase, fat was found in all sites. Leptin selectively breaks down fat stores.
Zhou et al. showed how enzymes for fatty acid oxidation are upregulated in response to leptin (1997). In cultured pancreatic islets, or hormone producing cells of pancreas, there was an upregulation of mRNA for fatty acid oxidation enzymes, acetyl CoA oxidase (ACO), and carnitine palmitoyl transferase (CPT-I) in leptin treated cells (Zhou et al, 1997).
mRNA ratio of enzymes for FFA metabolism to B actin in Zucker fa/fa rats. Acetyl CoA oxidase (ACO), and carnitine palmitoyl transferase (CPT-I), acetyl coA carboxylase (ACC) and glycerol-3-phosphate (GPAT) (Zhou et al., 1997).
A significant increase in ACO and CPT-1 in leptin infused fa/fa rats, two enzymes increasing FFA metabolism. A point of criticism for the mRNA concentrations are presented as a ratio to B actin. Perhaps leptin indirectly changed the mRNA concentration of B actin. If leptin lowered B actin mRNA, then the increased mRNA of fatty acid oxidation enzymes would be exaggerated, and thus less significant. However, numerous studies have been done confirming elevation of fatty acid oxidation enzymes.
Shimabukuro et al. (1997), showed how leptin esterifies FFA in adipocytes and nonadipocytes, stores them as TG, and later oxidizes them intracellularly. TG depletion of tissues has a direct antidiabetic effect on ZDF mice. Decreased fat stores helps maintain insulin sensitivity and prevents pancreatic islet lipotoxicity (Shimabukuro et al., 1997).
Fatty Acid Synthesis Suppression
Zhou et al. (1997) shows a decrease in mRNA for acetyl coA carboxylase (ACC) and glycerol-3-phosphate (GPAT) which are enzymes involved in biosynthesis of lipids. Similarly, Jiang et al. showed altered gene expression in WAT over 4 weeks of high fat diet fed mice (2009). Since high fat diet causes hyperleptinemia, the decreased concentration of lipogenic enzymes may be due to leptin action. With a sustained high fat diet for up to 16 weeks, altered gene expression occurred for adaptation to overnutrition (Jiang et al., 2009). Decreased lipogenic enzyme expression causes serum TG to be used as fuel, instead of proliferation of adipocytes for energy storage. Lipogenic enzymes were suppressed in white adipose tissue and in the liver. If all serum TG are not used as fuel, leptin directs serum triglycerides into intracellular lipid stores, instead of making new fat cells.
Intracellular Lipid Stores
Before evolution of adipocytes, each cell carried its own TG reservoir as an energy reserve. In time, adipocytes provided means for excessive energy storage, as fat cells multiply and increase in size with increased circulation of FFA (Spiegelman and Flier, 1996). Intracellular TG in nonadipocytes are still present, and may be regulated by leptin (Unger et al., 1999). When leptin receptors are dysfunctional, there was a 100-fold increase in TG in pancreatic islets, and
It has been shown that leptin decreases intracellular lipid storage, transporting the TG out of the cell to the FFA form. Shimabukuro et al. showed that the leptin-mediated increase in FFA was due to release of intracellular fat pools from adipocytes and nonadipocytes (1997). Using ZDF fa/fa rats, which are leptin receptor deficient, had decreased response to hyperleptinemia compared to controls. Therefore, regulation of intracellular lipid content is leptin receptor dependent (Shimabukuro et al., 1997).
Leptin and Triglycerides
Leptin signals to the brain the amount of triglyceride storage available. Triglycerides have been shown to inhibit leptin transport across the BBB. This paradoxical phenomenon may explain leptin resistance. One would assume that with increased triglycerides circulating in the body is a signal that the body is in a fed state. However, it is hypothesized that this mechanism of leptin evolved in the starvation state. High triglycerides, or hypertriglyceridemia is "a signal to the brain of starvation and not a signal of obesity" (Banks, 2006). The body assumes the triglycerides are from breakdown of body fat stores, therefore the signal to stop eating, or increase body fat metabolism is not necessary. Evolutionarily, more deaths come from starvation than obesity.
VIII. Pathology of Leptin
Exogenous leptin does not work in humans to cause weight loss, like it has been shown in mice (Pelleymounter et al, 1995). Due to the fact that obese and overweight individuals have chronic hyperleptinemia, there is an adaptive down-regulation of leptin responsiveness that leads to leptin resistance. With more fat present, more leptin circulates. Therefore, the body adjusts to a high level of leptin, and sensitivity to the signal decreases in the brain to decrease food intake. The mechanisms for down regulation of response to leptin are still being elucidated, as leptin resistance may be the key factor preventing pharmacological use of leptin in obese individuals.
The first question becomes, does leptin resistance occur in the CNS or periphery? In a study on diet-induced obese mice, resistance developed peripherally after 16 days of intraperitoneal leptin injections in mice on 45% fat diet; and, mice on 10% fat diet retained leptin-sensitivity (Van Heek et al., 1997). Peripherally leptin resistant AKR mice were given 10 and 45% fat diets and administered leptin peripherally. After 56 days, both groups became leptin-resistant. Central administration of leptin to normal and AKR mice showed continuous decreases in food intake, and no central resistance. This paper concluded that diet-induced obesity results in peripheral resistance, not central resistance (Van Heek et al., 1997). However, these results have been disputed.
Leptin is transported into the CNS by a saturable transport mechanism. With aid of OB-Ra receptor, leptin enters the CNS via endocytosis (Tu et al., 2010). Resistance may result from inhibition of leptin transport across the BBB. If leptin transport is Ob-Ra receptor dependent, then a mutation or down-regulation of OB-Ra may result in leptin resistance. Since the central mechanism of leptin acts through the hypothalamus, obstruction of transport would decrease leptin's metabolic effects.
Signaling mechanisms that may contribute to resistance is inhibition of JAK2, or up regulation of negative regulators SOCS3 and PTP1B (Morris and Rui, 2009). Another proposed mechanism of leptin resistance is ER stress (Morris and Rui, 2009). ER stress results from an accumulation of misfolded proteins in the ER lumen, which induces an uncoupled protein response (UPR). In intro, ER stress inhibits leptin signaling; however, pharmacological inhibition of ER stress enhances leptin response in vivo (Morris and Rui, 2009). The link between UPR and leptin signaling has not been clarified.
Congenital leptin deficiency is a rare disease characterized by early onset of obesity before the age of 10 years old. Other symptoms include, decreased CD4+ T cells and reproductive hormones, cold intolerance (Farooqi et al., 2002), as well as low brain weight, decreased DNA content in neurons, and impaired myelination (Ziylan et al., 2009). Leptin deficiency implies mutation in the leptin protein itself, not necessarily the receptor. Congenital leptin deficiency due to homozygosity of ?133G mutation has been identified in two Pakistani families from the UK, and a similar mutation in a Turkish family (Gibson et al., 2004). Although mutations in the leptin receptor are much more common than mutations in the protein itself, these congenital cases have proven valuable for analysis of leptin deficiency in vivo—a human version of the ob/ob mouse model.
In a study on 3 leptin deficient children—including two first cousins—exogenous recombinant leptin was administered daily for 4 years. Leptin therapy successfully increased gonadotropin secretion, which aided in timed sexual maturity. Weight loss occurred in the form of fat mass, and CD4+ T cell count increased (Farooqi et al., 2002). Overall, leptin deficiency is one of the only identified uses of leptin in humans to regulate energy homeostasis.
IX. Clinical Applications and Future Horizons
How can scientific knowledge of leptin be used to change human metabolism? The most applicable, relevant part of studying leptin, its molecular function, and its role in maintaining fat and energy balance, is to apply that knowledge clinically, and essentially, change metabolism.
Clinically, an upregulation of the leptin response may enhance fat loss humans. Prospective pharmacological uses of leptin include upregulation of synthesis, secretion, transcription, or receptor expression.
Leptin as a therapeutic agent
Initially, applying exogenous leptin was thought to diminish body weight in humans, similar to the way it did in rats (Halaas et al., 1995). However there are at least three reasons exogenous leptin administration does not work to diminish obesity in humans. Firstly, the majority of obese individuals are leptin resistant, in the CNS and/or peripherally. Secondly, pluripotent actions of leptin cause a multitude of side effects. Thirdly, leptin functions as a physiological sensor for energy stores: a signal for energy deprivation, not energy excess.
Another limiting factor for leptin as a therapeutic agent is its short half life. A Pluronic form of leptin (P85) was made to increase its circulating half-life by chemically modifying the structure by conjugating leptin with poly(ethylene oxide)- proly(propylene oxide) block copolymer (Price et al., 2010). This conjugation increases leptin circulation time by increasing stability and serum half-life, and decreasing elimination rate (Prince et al., 2010). When ICV injections of P85 were given to mice, there was a decrease in food intake, decreased clearance, and increased half life (from 5.46 min for leptin to 32.3 minutes). When P85 was radioactively labeled with iodine (125I), penetration of pluronic leptin into the BBB was measured. 125I-leptin crossed the BBB via a non saturable system, unrelated to the leptin transporter (Price et al., 2010). These studies show that a modified form of leptin could be developed for pharmacological use. While P85 was only tested on mice in this experiment, it maintained biological activity of leptin in vivo, giving hope for a structural analog of leptin safe for use in humans. If a modified form of leptin could be synthesized to only work on receptors in the CNS, it may be valuable pharmacologically to alter energy homeostasis.
Leptin and Diabetes
The latest studies on leptin and diabetes show leptin as progressive therapy for diabetes (Hedbacker et al., 2009). Direct application of leptin works to promote insulin sensitivity by transcriptional mechanisms. Leptin's enhancement of leptin sensitivity occurs via insulin growth factor binding protein2 (IGFBP2). Leptin regulates IGFBP2 gene expression, and caused a 3-fold improvement in hepatic insulin sensitivity after administration of IGFBP2 in ob/ob mice (Hedbacker et al., 2009). These new exciting studies provide an example of utilizing leptin signaling to alter energy metabolism clinically.
Leptin and nutrition
Since leptin electively enhances fat metabolism, does leptin help notify the body what kind of nutrients it needs? Does leptin cause nutrient specific cravings? Does it sense when there is a mineral, nutrient or macromolecule deficiency?
In a study on macronutrient preference, rats were centrally administered leptin until fat stores were completely depleted (Wiater et al., 2007). In this fat-depleted "nadir," food intake was monitored as the rats returned to normal body weight. Results show an increase in protein appetite.
Macronutrient selection in rats given daily leptin or aCSF injections for 8 days into the lateral cerebroventricle. Leptin treated rats were fat deficient before food presentation. Baseline measurements were taken during the 3 days before macronutrient selection test (Wiater et al., 2007).
Central leptin induced fat depleted rats showed significant preference for protein, and decreased preference for carbohydrate compared to aCSF control mice (Wiater et al., 2007). In a state of starvation, a protein appetite is favorable to combat muscle breakdown.
Vitamin supplements have been shown to enhance leptin expression. Retinoic acid, or vitamin A, has been shown to elevate expression of leptin, Glut 4, an ppar? in rat adipose tissue (Krskova-tybitanclova et al., 2008). Also, fish oil has been shown to enhance expression of leptin in adipose tissue (Selenscig et al., 2010).
While the diverse actions of leptin are still becoming elucidated, substantial progress has been made in the last 15 years. Obesity is now viewed as an endocrine disorder, with fat tissue as the primary endocrine organ. Leptin's regulation of energy metabolism may one day provide means to change human body weight and composition clinically.
Every individual has some "set point" body weight that can change throughout a lifetime. Aside from blatant cases of leptin deficiency, or leptin resistance, leading to obesity, how might leptin signaling genetically vary between individuals? Could an individual with more leptin receptors in the hypothalamus, or peripheral tissue be more responsive to leptin's action to decrease food intake and increase metabolism? May one person be able to eat as much fat as desired without gaining weight simply due to an up regulation of leptin receptors, or an enhancement of leptin crossing the BBB? Could the gene for leptin OB-Ra receptor become a key target for gene modification?
Why might individuals have different baseline for metabolism? The way a bird has a fast beating heart and a fast metabolism, versus a whale with a slow metabolism for a big creature. The animal model for leptin has been the ob/ob mouse; however, every organism is different, and marked differences in leptin action between mice and humans demonstrates the intimate complexity of this hormonal energy regulator. One aspect of genetics of fat metabolism becomes obvious with studying different geographic populations: genetic variations in metabolism arising from geographic differences. By studying variations in leptin response between individuals, there may be a way to modify genetic weight predispositions clinically.
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Leptin is a 16kD hormone secreted by adipose tissue that regulates a variety of homeostatic mechanisms—principally, energy metabolism. Leptin acts peripherally and centrally to regulate food intake, lipid metabolism and energy homeostasis. Leptin has short term effects to increase fat metabolism, and long term effects to decrease body weight. The discovery of leptin arose from analysis of the ob gene from obese mouse model, ob/ob. Administration of OB gene product to ob/ob mice resulted in selective fat breakdown, increased body temperature, and weight loss. The db (diabetes) gene, isolated from db/db mice codes for the leptin receptor. There are at least 5 isoforms of the Ob receptor, only one with signal transduction capability (OB-Rb). Leptin signaling involves the JAK/STAT pathway and activates a variety of intracellular signaling pathways including MAPK, AMPK, and mTOR to alter gene expression and enhance fat metabolism. Centrally, leptin acts on receptors on the hypothalamus to activate anorexigenic neurons (POMC), and inhibit orexigenic neurons (AgRP/NPY), to increase appetite, increase temperature and SNS outflow, and modulate selection of fuel metabolism. In peripheral tissue, leptin incites an increase in lipid metabolism by elevating levels of fatty acid oxidation enzymes, decreasing lipid synthesis, and intracellular fat stores in nonadipocytes. As a signal for adiposity, serum leptin levels notify the brain of available energy stores. It is proposed that leptin evolved as an adaptation to starvation, not energy excess. Low leptin levels in starvation produce an increased protein appetite, decreased reproductive function, and preservation of fat stores. Therefore, chronically elevated levels of leptin in obese individuals leads to down regulation of leptin response and, in many cases, leptin resistance. Leptin resistance illustrates why exogenous leptin does not work to diminish body weight in human obesity. The mechanisms of leptin resistance are unclear. Clinically, leptin can be used in congenital leptin deficiency, anorexic amenorrhea, however does not alter energy homeostasis in obese humans. Recent studies show leptin may be used to treat diabetes in humans, because leptin enhances insulin-sensitivity. Through elucidation of leptin signaling, a pharmacological agent may be developed to enhance its weight-reducing effect.