LepR LOF lines - are they bona fide?

Evidence suggests that fish with a loss of function in leptin a or the leptin receptor are obese or not depending on the paper one reads and it has been suggested that maybe the Lepr sa1508 line of zebrafish is potentially a hypomorph and therefore does not express obesity. We sat out to a) produce more Lepr and LepA alleles in which this can be tested in our hands and b) test whether leptin signalling was abolished in our Lepr LOF lines. 

For this we decided to inject mammalian leptin (which is structurally conserved to fish leptin and we do not have a good source of homologous leptin yet) and see whether we would find the immediate early gene SOCS3a to be upregulated. We found that indeed zebrafish livers respond t IP leptin injections with an upregulation of SOCS3a. Importantly, this effect was specifically abolished in all of three Lepr LOF lines we have, sa1508 and two lines we made using CRISPR Cas9. Therefore, at least as far as the SOCS signal transduction pathway downstream of the Lepr is concerned, these three Lepr LOF lines are bona fide loss of function. - (Bagivalu Lakshminarasimha et al., 2021)

Some caveats to fish growth experiments.

We further embarked to see whether maybe our setup cause some of the effects, namely I had early in my career found significantly enhanced growth in a line which was abolished upon raising the line as a het incross. Often, zebrafish lines are crossed as -/- x -/- either with a sibling wildtype incross and/or with simple wildtype controls which are kept as a different stock. This as a practice may be fine when using mouse lines which are incredibly inbred and show very little to no variation along the genome. Zebrafish instead are very outbred and show similar or more variability in the genome as humans. Further, growth in fish is very dependent on food levels and density of fish in the habitat they are in so even slight variation between tanks in the number of fish or the food given can lead to differences in growth.

When we controlled for everything by raising a het x het cross and separated the fish at 3 months of age, scored their weight and length and then genotyped the fish we found again no differences in growth amongst these lines. When we however raised a crossing of +/+  x +/+ separate to a cross of -/- x -/- we found slight differences in growth at 3 months of age which likely would amplify if the fish were allowed to grow further. These differences - some bigger some smaller did not corellate with the genotype however showing that Lepr or lep LOF did not drive these differences. - (Bagivalu Lakshminarasimha et al., 2021)

Fish growth - do zebrafish become obese?

One question that arises when seeing that leptin or lepr LOF animals are NOT obese - do zebrafish get obese?

It has of course been published that overfed zebraifsh deposit more fat and that transcriptionally (Oka et al., 2010). The authors also showed increased lipid accumulation in the liver, increase plasma triglycerides as well as conserved expression profiles between visceral fish adipose and mammalian adipose. However, leptin, a main adipose expressed and secreted gene is not present in zebrafish adipose!

Therefore we overfed fish from day 5 onwards on a long-term obesogenic diet and compared this to calorically restricted fish. Further we allowed calorically restricted fish to compensate at three timepoints of growth, early in their life (1 month during which they still grow exponentially), at three months during which time they grow linearly and at 9 moths during which time control fish growth plateaus out. We hypothesized that early food restriction would exacerbate obesity endophenotypes.

We found that at all three timepoints zebrafish caught up and did not undergo compensatory growth according to Jobling (2010), that is fish growth rate in the group catching up never increased above and beyond what a fish normally developing on an obesogenic diet would. However, the weight-length relationship of zebrafish on the obesogenic diet clearly showed positive allometric growth meaning that fish at first grow in length and later as they age and become longer they grow relatively in circumference and not only in length, i.e. obese. We similarly found that young fish grow primarily in length and later gain significant body fat deposits as they age and slowly stop growing (i.e. hit a growth plateau for standard length). We found evidence for hepatosteatosis and hyperglycemia in the obese fish, however overall the obese fish were largely healthy. - (Leibold et al., 2022)

In some ways this study opened more questions than it answered. If fish adipose deposition differs during ageing - is it critical when lepr LOF fish are tested for adiposity? Mice and people of course show signs of severe early onset morbid obesity, fish do not. However Herman Spainks group showed that leptin b LOF fish become obese at 1-2 years of age (He et al., 2021). How does fat deposition change as fish age? Of course, this is a know phenomenon in people.

Further - what are comorbidities or endophenotypes of obesity? Metabolic syndrom encompasses a lot of co-morbidities but the problem is penetration - not all people get these comorbidities. We have thus far not looked at strain differences between zebrafish strains. In one recent study, Helene Volkoff did look at two different strains of zebraifsh and found that expression patterns of energy homeostasis genes varied quite considerably (London and Volkoff, 2019).

The role of leptin in fish reproductive biology

Wile characterizing the Agrp and Lepr LOF lines we found that these fish had pretty serious drops in pituitary gene expression, paricularly in the reproductive axis (fsh, lh). However we did not find overt breeding phenotypes. This result stands in stark contrast with data from Yonathan Zohars lab showing that Gnrh LOF fish also have no overt reproductive phenotype but also have normal pituitary gene expression, suggesting central compensation (Marvel et al. 2018 & Marvel et al. 2019). In our case, we also see no reproductive phenotype but a drop of central gene expression. In the Lepr LOF fish we have so far found that while fish breed fine when netted out of the tanks, they show a delay in maturation when all oocytes are first purged by one or two bouts of breeding in a mating cage which in our hands leads to the release of nearly all mature oocytes by the female. When we looked at oocyte maturation after such an even we found that oocytes mature in a delayed manner and that oocytes show signs of atresia a lot earlier in Lepr LOF fish. Atresia is the mechanism of resorption of oocytes and it's biological relevance is not entirely clear but it could be a mechanism to keep oocytes relatively fresh until a mating opportunity arises for the individual fish or it could be a means of energy resorption. This process occurs naturally when female zebrafish are separated from males. For about 8 days, oocytes mature and the gonadosomatic index reaches a plateau, at which time a slow cycle of continuous resorption and maturation starts. Lepr LOF females start this process earlier around 4 days and it is so far unclear whether this is due to a delay in maturation or an earlier initiation of atresia or both. Further, we showed using in vitro assay of germinal vesicle breakdown (GVBD) that exogenous leptin and Lepr LOF have an effect on GVBD, that is the last step in ovarian maturation. - (Bagivalu Lakshminarasimha et al., 2022).

Parallel to our study, Monika Schmitz showed in her lab that Lepr LOF animals for a different allele are subfertile and perform considerable poorer than wildtype controls. She further shows that this effect can be overridden by injection of human chorionic gonadotropin (hCG), suggesting that the effect is central (Tsakoumis et al., 2022).