Assignment 4 - Critique of a Recent Article About Leptin

“Relationship between plasma leptin-like protein levels, begging and provisioning in nestling thin-billed prions Pachyptila belcheri”

Quillfeldt, P., Everaert, N., Buyse, J., Masello, J.F., and Dridi, S. (2009). General and Comparative Endocrinology. 161 171-178. Available Online

The thin-billed prion, Pachyptila belcheri (taken from Wikipedia)

Summary:

                In this paper published in 2009, Quillfeldt et al. present the first study conducted to analyze the physiological role of leptin in free living seabirds. The species chosen, Pachyptila belcheri, is a procellariiform (“tubenosed”) seabird, known to breed many times during its life cycle but only raise one chick at a time. The chicks are fed infrequently and at irregular time intervals, but despite this are observed to grow rapidly and accumulate large amounts of lipid as nestlings. Because of this characteristic, termed “nestling obesity,” this species appears to be a good choice in which to investigate the possible role of leptin in sensing of body energy reserves and feeding associated behavour in seabirds.

                An interesting controversy is ongoing as to the existence of a leptin gene in bird species, particularly the chicken – the cloning and sequencing of a chicken gene was reported but the validity of the data was later challenged. As such, the Quillfeldt et al. refer to the protein analyzed in P. belcheri as “leptin-like protein” to acknowledge the possibility of it being another closely related protein. In other bird species, leptin-like protein is seen to be expressed not only in the adipose tissue but also in the liver, which is likely related to the major role of the liver in lipogenesis regulation in birds. Quillfeldt et al. thus had two main goals in this study: to determine whether leptin is expressed in liver and adipose tissue of thin-billed prions (P. belcheri); and to investigate the relationship between plasma leptin levels, body conditions, provisioning rate, and begging intensity.

                The study was carried out over two nesting seasons at New Island, Falkland Islands. P. belcheri chick specimens were weighed twice daily to produce an index of body condition, while tape recorders were used to collect data on the chicks’ begging behavior (calling for food when adults return to the nest) – both indicators of the nutritional state of the specimens. Blood samples were taken for the analysis of plasma glucose, triglyceride, and leptin-like protein levels. The latter was measured by radioimmunoassay, while the other two were measured spectrophotometrically. Statistical analysis of the data was performed using the “SPS 12.0.1” program. Finally, a Western blot analysis was performed using liver and adipose tissue from a deceased specimen, in which leptin-like protein was detected using rabbit polyclonal anti-leptin antibody and goat anti-rabbit secondary antibody.

Results:

1)      The Western blot (Figure 1 in paper) indicated a band at approximately 14-16 kDa (leptin is about 16kDa) for both liver and adipose tissue, consistent with the positive control of chicken liver, indicating the presence of a leptin-like protein.

2)      Plasma leptin-like protein levels in the chicks was between 1 and 3 ng/ml, similar to that seen in chickens. These levels did not correlate with plasma triglyceride or plasma glucose levels.

3)      No correlation between leptin-like protein levels and body condition was observed (Table 1 in paper), but levels were seen to negatively correlate with the provisioning rate (leptin-like protein was lower as chicks were fed more often.)

4)      A few cases of elevated (above 3.2 ng/ml) leptin-like protein were observed, which were associated with elevated plasma triglyceride levels (Figure 5 in paper).

5)      Plasma leptin-like protein levels were positively correlated to begging call number, although begging intensity was not correlated to provisioning rate or body condition (Table 1 in paper).

6)      Plasma leptin-like protein levels did not differ between the two seasons, but were observed to increase with age of the chicks (Figure 7 in paper).

These results are interesting, particularly # 3 and #5, as they indicate that chicks that were fed less often and begged for food more incessantly had higher levels of leptin-like protein. This is in contrast to most studies of leptin, where high levels correlate with high levels of body fat and thus a fed state. Leptin-like protein levels were not proportional to triglyceride levels in P. belcheri ; the authors speculate this may be related to production of the protein by the liver, and a particular role for leptin in lipid metabolism in birds.

Critique:

• The paper was overall well written, and was a good length (not too long but didn’t seem as though things were left out). In some places the wording/grammar seemed a little off, but this may be due to a language issue as the authors are from Germany and Belgium.

• Most of the problems with this study are in fact pointed out by the authors in their discussion, including the main issue that the protein studied is not conclusively confirmed as leptin (a protein very similar to leptin could have been measured by the radioimunnoassay).

• Quillfeldt et al. admit that the correlation seen between leptin-like protein levels and begging intensities is unexpected and the reason for the correlation is unclear. They try to propose a possible explanation, but it is worded poorly and is difficult to understand.

• They also admit that their method of measuring leptin-like protein concentration does not take into account dinural patterns of leptin concentrations, which are known to occur in other species.

Future Experiments:

• First and most importantly, the gene for the leptin-like protein must be isolated sequenced using cloning techniques, so that it can be compared to known leptin sequences.

 • Studies on the effect of recombinant leptin introduced into P. belcheri would be useful to confirm if a relationship exists between leptin levels and triglyceride levels, following up on the observations recorded for individuals with above average leptin-like protein levels.

• Studies using similar methods could be done using closely related bird species, to see if the unusual results observed in this study are repeated.

Assignment 3 - Leptin Function

Leptin and Obesity:

From its discovery in obese mice, it was clear that the hormone leptin had a function in metabolism and energy homeostasis. The lipostasis theory of energy balance had already predicted that the amount of fat stored by the body was regulated by the central nervous system based on the action of a circulating product of fat metabolism on the hypothalamus (Zhang et al., 1994). The isolation of the product of the obese gene, later named leptin, provided validation for this theory. Leptin is sometimes called an “adipostat,” as it signals the status of the body’s energy stores to the brain and thus acts in metabolic regulation (Houseknecht and Portocarrero, 1998). Generally, the level of circulating leptin indicates the amount of energy stored as fat in the body.

Leptin acts through binding leptin receptors, known as OB-R. One isoform of this receptor (OB-Ra) is found in many different tissues, including the lung, kidney, liver, and B cells of the pancreas (reviewed in Houseknecht and Portocarrero, 1998). Another isoform (OB-Rb) is predominantly expressed in the ventromedial hypothalamus, a region of the brain known to be important in energy homeostasis, and particularly appetite regulation. When leptin binds OB-R, two receptors dimerize and can activate a Jak-Stat signaling pathway, or other signaling pathways, leading to the activation of a complex pathway of appetite enhancing and suppressing neuropeptides to adjust food intake (Houseknecht and Portocarrero, 1998; Kelesidis et al., 2010). For example, leptin inhibits the synthesis of neuropeptide Y (NPY), a peptide which stimulates appetite and is often increased in models of obesity (Stephens et al., 1995).

Figure 1: Leptin / leptin receptor mechanism of action. Originally from Houseknecht and Portocarrero, 1998.

A homozygous mutation in the leptin gene results in extreme obesity in both mice and humans. The leptin deficient ob/ob mice were observed to overeat (hyperphagia), and to have neuroendocrine abnormalities and diabetes, and be infertile (reviewed in Kelesidis et al., 2010). A similar pathology is seen in human patients with congenital leptin deficiency. Treatment with recombinant leptin is very effective in such patients, causing a reduction in weight due to decreased appetite, which highlights the importance of leptin in regulating food intake (Farooqi et al., 1999; Kelesidis et al., 2010). However, as leptin deficiency accounts for only a small percentage of obesity in humans, the hormone does not give us an ‘obesity cure’ as was originally hoped upon its discovery. Obesity instead is usually due to a resistance or intolerance to leptin, likely as a result of defective OB-R, and patients in fact have higher than normal leptin levels correlating to a larger mass of adipose tissue (Houseknecht and Portocarrero 1998; Kelesidis et al., 2010). In both cases, the hypothalamus does not receive any signal to indicate that the body has sufficient stores of fat, so no suppression of appetite occurs.

Leptin as more than just the “obese gene”:

Recent studies have shown that the more important role of leptin is to indicate energy deficiency, rather than to prevent obesity (Kelesidis et al., 2010). During fasting, levels of the hormone drop drastically and very quickly. This induces a neuroendocrine response which includes a decrease in gonadotropins and sex steroids, decreasing fertility; as well as a decrease in thyroxine levels, slowing metabolism (Ahima et al., 1996). Leptin injection during starvation has been shown to alleviate these effects.Understanding this role of leptin has led to the finding that hypoleptinemia (decreased leptin) is associated with anorexia nervosa and hypothalamic amenorrhea (cessation of menstruation, often due to strenuous exercise), both of which are characterized by decreased body fat (Kelesidis et al., 2010). Trials have indicated that leptin treatment of hypothalamic amenorrhea is quite effective, restoring levels of estrogen, thyroxine and IGF1, and restoring menstruation (Welt et al., 2004). Effects on markers of bone formation were also observed, indicating a role of leptin in that process.

Figure 2: Effects of leptin and leptin deficiency in both overfed (energy excess) and underfed (energy deficiency) states. Originally from Kelesidis et al., 2010)

. . . Of course the above only gives a partial list of the actions and interactions of leptin. A complete description of the roles of leptin in energy homeostasis would take far more space than can be given here!


References:
Ahima, R.S., Prabakaran, D., Mantzoros, C., Qu, D., Lowell, B., Maratos-Flier E., and Flier, J.S. (1996). Role of leptin in the neuroendocrine response to fasting. Nature 382(6588) 250-252.

Farooqi, I.S., Jebb, S.A., Langmack, G., Lawerence, E., Cheetham, C.H., Prentice, A.M., et al. (1999). Effects of recombinant leptin therapy in a child with congenital leptin deficiency. New England Journal of Medicine. 341 879-884.

Houseknecht, K.L., and Portocarrero, C.P. (1998). Leptin and its receptors: regulators of whole-body energy homeostasis. Domestic Animal Endocrinology 15(6) 457-475.

Kelesidis, T., Kelesidis, I., Chou, S., and Mantzoros, C.S. (2010). Narrative review: the role of leptin in human physiology: emerging clinical applications. Annals of Internal Medicine 152(2) 93-100.

Stephens, T.W., Basinski, M., Bristow, P.K., Bue-Valleskey, J.M., Burgett, S.G., Craft, L., et al. (1995). The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature 377(6549) 530-532.

Welt, C.K., Chan, J.L., Bullen, J., Murphy, R., Smith, P., DePaoli, A.M., Karalis, A., and Mantzoros, C.S. (2004). Recombinant human leptin in women with hypothalamic amenorrhea. New England Journal of Medicine 351(10) 987-997.

Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., and Friedman, J.M. (1994). Positional cloning of the mouse obese gene and its human homologue. Nature 372(6505) 425-32.

 

Assignment 2 - Leptin Structure

Figure 1: Crystal Structure of Leptin. (Taken from NCBI website).

The human leptin gene is just over 16,000 base pairs in length, and is located on chromosome 7, at 7q31.3 (NCBI). It encodes a 16 kDa, 167 amino acid protein with four antiparallel alpha helices, similar to the long-chain helical cytokine family of proteins (Zhang et al., 2007). The tertiary structure of the protein includes a "hydrophobic core" of amino acids facing inward on each helix, and a disulphide bond between cysteine residues 96 and 146. This disulphide bond is particularly important to folding of the protein and for binding the leptin receptor (Fruhbeck, 2006). As seen in the protein alignment below, Cys96 and Cys146 are conserved between species as varied as human, chicken and sturgeon. Sections of the four alpha helices are also well conserved between species (Zhang et al., 2007).

Figure 1: Alignment of protein sequences for Mus musculus leptin

 (Accession number NM_008493), Gallus gallus leptin (AF012727), Acipenser

 schrenckii leptin (DQ784816) and Homo sapiens leptin (U43653). Prepared using

 ClustalW software.

 

The scores associated with this alignment indicate that the most similar sequences are those from the mouse and sturgeon, while the human sequence is least similar to any of the others.

Table 1: Alignment Scores for Mouse, Human, Chicken and Sturgeon

Leptin Protein Sequences

SeqA Name       Len(aa)  SeqB Name       Len(aa)  Score

=======================================================

1    mouse      167      2    human      167      83   

1    mouse      167      3    chicken    163      96   

1    mouse      167      4    sturgeon   146      99   

2    human      167      3    chicken    163      80   

2    human      167      4    sturgeon   146      84   

3    chicken    163      4    sturgeon   146      95   

=======================================================

Assignment 1 - Leptin (My Favourite Hormone)

Leptin is a 167 amino acid, 16kb protein hormone which is synthesized and secreted by adipose tissue.  It is also produced in lesser amounts in tissues such as the placenta, ovaries, mammary epithelium, and bone marrow (Dardeno et al., 2010). Its levels within the body follow a circadian rhythm, peaking late at night and decreasing during the day. Leptin secretion has been shown to be affected by sex hormone levels, with women generally having more of the hormone than men (reviewed in Dardeno et al., 2010).

Figure 1: Normal mouse (left) and ob/ob mouse. Taken from "Genome News Network"

Leptin is the product of the obese (ob) gene, originally studied in mice, where a homozygous mutation (ob/ob) was known to cause obesity (Dardeno et al., 2010). It was unknown how this mutation caused obesity; however, until 1994 when Zhang et al. cloned both the mouse and human ob genes (Zhang et al., 1994; reviewed in Dardeno et al., 2010). They discovered that the gene product was a secreted adipose tissue protein, highly conserved between mice and humans, and important in regulating energy balance (Zhang et al., 1994). This protein was later determined to be a hormone, and named leptin, from the Greek root leptόs, meaning “thin,” as it had been shown to reduce the weight of obese mice (Halaas et al., 1995). 

Leptin is an important regulator of the amount of fat stored within the body. It can cross the blood-brain barrier, and acts on different areas of the hypothalamus to stimulate energy expenditure and suppress appetite (Kiess et al., 2008). It has also been shown that leptin acts on areas of the brain that control arousal, mood, and reward, decreasing an animal’s desire for food, while roles in brain development, immune function, and bone metabolism have also been demonstrated (reviewed in Dardeno et al., 2010).

             The leptin receptor (Ob-R) has several isoforms, which are classified as long, short and soluble. These receptors are found throughout the central nervous system, but also in various tissues such as the kidney, liver, intestine, stomach and heart (Kiess et al., 2008). When leptin binds its receptor, several signaling pathways are activated, including the Jak2/Stat3, PI3K, and MAPK pathways, leading to the observed effects on appetite and body weight (Dardeno et al., 2010).

Although it has been shown that leptin cannot simply cure obesity (obese humans are generally resistant to the hormone), ongoing research with leptin and leptin sensitizers may lead to therapies for weight loss and weight loss maintenance in the future.

References:
Dardeno, T.A., Chou, S.H., Moon, H.S., Chamberland, J.P., Fiorenza, C.G., and Mantzoros, C.S. (2010). Leptin in human physiology and therapeutics. Frontiers in Neuroendocrinology 31 377-93.
Halaas, J.L., Gajiwala K.S., Maffei M., Cohen S.L., Chait B.T., Rabinowitz D., Lallone R.L., Burley S.K., Friedman J.M. (1995). Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269(5223) 543-6.
Kiess, W., Petzold, S., Töpfer, M., Garten, A., Blüher, S., Kapellen, T., Körner, A., Kratzsch, J. (2008). Adipocytes and adipose tissue. Best Practice and Research Clinical Endocrinology and Metabolism 22 135-53.
Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., and Friedman, J.M. (1994). Positional cloning of the mouse obese gene and its human homologue. Nature 372(6505) 425-32.