Leptin deficiency affects glucose homeostasis and results in adiposity in zebrafish

in Journal of Endocrinology
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  • 1 Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands
  • 2 Department of Animal Sciences and Health, Institute of Biology, Leiden University, Leiden, The Netherlands
  • 3 Department of Animal Anatomy, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
  • 4 Institute of Medical Physics and Biophysics, University of Leipzig, Leipzig, Germany
  • 5 Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands

Correspondence should be addressed to H P Spaink: h.p.spaink@biology.leidenuniv.nl

*(J He and Y Ding contributed equally to this work)

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Leptin is a hormone which functions in the regulation of energy homeostasis via suppression of appetite. In zebrafish, there are two paralogous genes encoding leptin, called lepa and lepb. In a gene expression study, we found that the lepb gene, not the lepa gene, was significantly downregulated under the state of insulin-resistance in zebrafish larvae, suggesting that the lepb plays a role in glucose homeostasis. In the current study, we characterised lepb-deficient (lepb−/−) adult zebrafish generated via a CRISPR-CAS9 gene editing approach by investigating whether the disruption of the lepb gene would result in the development of type 2 diabetes mellitus (T2DM) and diabetic complications. We observed that lepb−/− adult zebrafish had an increase in body weight, length and visceral fat accumulation, compared to age-matched control zebrafish. In addition, lepb−/− zebrafish had significantly higher blood glucose levels compared to control zebrafish. These data collectively indicate that lepb−/− adult zebrafish display the features of T2DM. Furthermore, we showed that lepb−/− adult zebrafish had glomerular hypertrophy and thickening of the glomerular basement membrane, compared to control zebrafish, suggesting that lepb−/− adult zebrafish develop early signs of diabetic nephropathy. In conclusion, our results demonstrate that lepb regulates glucose homeostasis and adiposity in zebrafish, and suggest that lepb−/− mutant zebrafish are a promising model to investigate the role of leptin in the development of T2DM and are an attractive model to perform mechanistic and therapeutic research in T2DM and its complications.

Supplementary Materials

    • Supplementary Figure 1. Lepb-deficient zebrafish line (A) The wild type zebrafish (Ctr) is showing a partial sequence of the lepb gene. Seven base pairs (TAGAGGG) were deleted in lepb7-/7- zebrafish and eight base pairs (TAGAGGGC) were deleted in lepb8-/8- zebrafish. The arrowheads show the start point of the deletion. (B) The wild type lepb gene translates into the predicted 19.1 kDa lepb protein in zebrafish. The seven base pairs deletion creates a premature stop codon UAG in exon 2, which results in a truncated 16.5 kDa protein. And the eight base pairs deletion causes a premature stop codon UAA in exon 2, which results in a truncated 13.3 kDa protein. (C) Homozygous F1 carriers were outcrossed once against the wild type zebrafish (ABTL) and the offspring (heterozygote lepb+/-) were subsequently incrossed, resulting in the used lepb+/+ and lepb-/- siblings. Because of the limitations of the number of adult zebrafish during this study, we used adult WT-ABTL (Ctr) and all lepb-/- (lepb7-/7-, lepb8-/8- and lepb7-/8-) for the experiments.
    • Supplementary Figure 2. Blood glucose levels in lepb+/+ adult zebrafish and age-matched ABTL control zebrafish Two hours postprandial blood glucose levels in ABTL control and lepb+/+ zebrafish (female and male). Ctr: ABTL control zebrafish; lepb+: lepb+/+ zebrafish; F: female; M: male.
    • Supplementary Figure 3. Body weight, length, BMI and blood glucose levels in different lepb-/- mutants (A) The body weight of control and lepb-/- female (**p<0.01) and male (*p<0.05) adult zebrafish. (B) The body length of control and lepb-/- female (*p<0.05) and male (**p<0.01) adult zebrafish. (C) The BMI of control and lepb-/- female and male adult zebrafish. (D) Two hours postprandial blood glucose levels in control and lepb-/- female and male (*p<0.05) adult zebrafish. Different lepb-/- mutants were marked in different colors. lepb7-/7- in red, lepb8-/8- in blue and lepb7-/8- in green. Ctr: control zebrafish; lepb-: lepb-/- zebrafish; F: female; M: male.
    • Supplementary Figure 4. Fasting blood glucose levels in control and lepb-deficient adult zebrafish Fasting blood glucose levels in control and lepb-/- female and male (*p<0.05) adult zebrafish. Ctr: control zebrafish; lepb-: lepb-/- zebrafish; F: female; M: male.

 

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