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Leptin Controls Glucose Homeostasis but not Obesity in Zebrafish

By: Carol A. Rouzer, VICB Communications
Published:  March 18, 2016


Zebrafish bearing a homozygous mutation of the leptin receptor exhibit altered glucose tolerance, but not the extreme obesity observed in mice bearing the same mutation.


In 1950, A. M. Ingalls and coworkers reported their discovery of a mutation that caused unusual obesity in the house mouse (J. Hered. 41, 317). These mice, designated ob/ob (Figure 1) became the focus of intense study over the ensuing years, as the human obesity epidemic became a growing public health problem. It was not until 1994, however, that Jeffrey Friedman and his colleagues reported the identity of the mutation responsible for this condition (Nature, 372, 425). The protein encoded by the mouse obese gene, ultimately named leptin, is a cytokine that is produced by adipocytes in mice. Leptin acts on receptors in the central nervous system to control feeding behavior, metabolism, and endocrine function. Mutations of the gene for leptin or its receptor in mice or humans lead to morbid obesity, excessive food intake, diabetes, slow metabolism, and poor fertility. Among mammals, the genes for both leptin and its receptor are highly conserved, but large disparities exist between the mammalian genes and those of lower vertebrates. For example the zebrafish (Danio rerio) and human leptin proteins are only 19% identical, although they retain comparable structural features and employ similar intracellular signaling pathways. Other major differences occur in expression profile. Whereas leptin secretion occurs predominantly in adipose tissue among mammals, in fish the primary source appears to be the liver. Remarkably, prior reports show increasing leptin levels with fasting in fish, the opposite pattern from that observed in mammals. These observations suggest that leptin’s role in lower vertebrates may differ from that in mammals. Now, Vanderbilt Institute of Chemical Biology members Roger Cone and Wenbiao Chen and their laboratories report new findings that support this hypothesis [M. Michel, et al. (2016) Proc. Natl. Acad. Sci. U.S.A., published online February 22, DOI:10.1073/pnas.1513212113].



FIGURE 1. Figure 1. An ob/ob mouse bearing a homozygous mutation of the Lep gene (right), and its normal sibling (left). Figure reprinted by permission from Macmillan Publishers LTD from R.L. Leibel (2008) Int. J. Obes. 32, S98. Copyright 2008.

The investigators focused their study on leptin function in zebrafish, obtaining a line bearing a mutation (sa1508) in the gene for the leptin receptor (lepr) that results in a truncated, nonfunctional protein. A similar mutation of the gene for the leptin receptor in mice produces a phenotype that is nearly identical to that of mice bearing a mutation in the gene for leptin. However, when the researchers compared homozygous leprsa1508/sa1508 zebrafish to wild-type siblings, they observed no differences in weight, length, body fat, feeding behavior, response to overfeeding, or fertility. Thus, the mutant zebrafish exhibited none of the phenotypic characteristics observed in mice with a comparable mutation.


Mice bearing a mutation in the gene for the leptin receptor exhibit hyperglycemia by three to four weeks of age, an indication of the importance of leptin’s role in glucose homeostasis. Thus, the investigators tested the hypothesis that leptin receptor deficiency would lead to abnormal glucose regulation in the zebrafish. They found a small but significant increase in the glucose content of leprsa1508/sa1508 as compared to wild-type zebrafish fry. The mutant fry also exhibited increased expression of the mRNA for insulin a (insa) but not insulin b (insb). In addition, the leprsa1508/sa1508 mutation led to increased mRNA expression of both glucagon genes (gcga and gcgb) in zebrafish fry. Increased expression of genes encoding proteins involved in glucose metabolism, including mitochondrial phosphoenolpyruvate carboxykinase (pck2), glycogen phosphorylase (pygl), glucose 6-phosphatase (g6pase), and glucose transporters (slc2a2 and slc2a5) suggested that the leprsa1508/sa1508 mutation resulted in an upregulation of glucose biosynthesis (gluconeogenesis), glucose release from glycogen (glycogenolysis), and some aspects of glucose transport. In contrast, the mutation had no effect on the expression of genes encoding pyruvate kinase (pklr), cytosolic phosphoenolpyruvate carboxykinase (pck1), or other glucose transporters (slc2a8 and slc2a9l1).


To further investigate the effects of the leprsa1508/sa1508 genotype on glucose homeostasis, the researchers crossed the sa1508 allele into mice bearing a transgene encoding a fluorescent β-cell marker. The resulting mutant fish enabled them to visualize the effects of the leptin receptor mutation on insulin-secreting cells in situ (Figure 2). The investigators discovered that the leprsa1508/sa1508 genotype resulted in a 25% increase in β-cell number. To confirm this finding, they used the CRISPR/cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9) gene editing system to knockout the two zebrafish genes encoding leptin proteins (lepa and lepb) and lepr in zebrafish bearing the β-cell marker transgene. They found that deletion of lepa and lepr resulted in increased β-cell numbers in zebrafish fry. This was not observed upon knockout of lepb or appropriate unrelated control genes. These results confirmed that a deficiency in leptin signaling results in increased β cells, possibly to compensate for the observed alterations in glucose homeostasis in the leprsa1508/sa1508 mutant fry. Consistent with this hypothesis, treatment with metformin, an inhibitor of hepatic gluconeogenesis, prevented the elevation in β-cell number in leprsa1508/sa1508 zebrafish.


FIGURE 2. Image of a zebrafish carrying the β-cell marker transgene (left), and a close-up photomicrograph of a cluster of fluorescent β-cells (right). Figure reproduced with permission from M. Michel, et al. (2016) Proc. Natl. Acad. Sci. U.S.A., published online February 22, DOI:10.1073/pnas.1513212113. Copyright 2016, M. Michel, et al.



In adult zebrafish, the investigators observed no difference in fasting glucose levels between leprsa1508/sa1508 mutants and their wild-type siblings. However, exposure of the fish to a glucose challenge resulted in a more rapid return to normal levels in the mutants than in the wild-type controls. Similarly, the mutant fish exhibited a higher increase in expression of insa and gcga following a meal than did their wild-type siblings. The adult leprsa1508/sa1508 mutants retained increased expression of the mRNAs encoding glycogen phosphorylase and two glucose transporters (slc2a5 and slc2a9l1), but levels of mRNAs encoding other enzymes of glucose homeostasis were no different from those in wild-type adults. Thus, the leprsa1508/sa1508 mutants did not display a diabetic phenotype; however the mutation was associated with a decreased capacity for wound healing, a characteristic associated with diabetes.


Prior research had demonstrated that exposure of wild-type zebrafish larvae to nutrient excess results in an increase in β-cell number that is driven by the secretion of fibroblast growth factor (FGF) from β cells. In contrast, the investigators found that exposure of leprsa1508/sa1508 mutant zebrafish to excess nutrients resulted in no further increase in β-cell number. Furthermore, exposure of the mutants to an inhibitor of FGF signaling resulted in a reduction in β-cell number to that of wild-type larvae. Thus, it appears that the increase in β-cells associated with a failure of leptin signaling is mediated, at least in part, by FGF.


The results suggest that, in zebrafish, the role of leptin is largely limited to modulation of glucose homeostasis. The investigators note that a similar phenotype is observed in mice bearing a leptin receptor knockout mutation that is restricted to the liver. These mice demonstrate an increase in β-cell number, normal fasting glucose levels, and increased glucose tolerance. These findings highlight an important function of leptin signaling that, in mammals, is overshadowed by its regulation of adipose tissue metabolism. Further work will be required to fully appreciate this role of leptin in lower vertebrate species and mammals.



View PNAS article: Leptin signaling regulates glucose homeostasis, but not adipostasis, in the zebrafish






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