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.