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Research - background

Energy homeostasis balances food intake with energy expenditure. We use energy via our daily activity (food seeking, general movement but also growth, reproduction et cetera), but also via the basal metabolic rate (the energy it takes to keep the body functioning in a resting state) and the thermic effect of food (the energy it takes to metabolize food and make its nutrients and energy available).

When this system is out of balance, metabolic diseases follow which collectively pose a huge societal burden. Obesity follows from an increased hunger drive, while a lack of appetite leads to anorexia and cancer cachexia. Each of these is comorbid with a variety of conditions, predominantly type 2 diabetes with obesity, but reproductive issues are also comorbid with both ends of the scale - obesity as well as anorexia. Therefore a good balance between food intake and expenditure is critical.

One might posit that eating too little or too much is largely a cultural and behavioural burden - too much high-calorie food, too little exercise whether due to increase work times or decreased interest; and these undoubtedly have a large impact. However, our genetics have a surprisingly large effect on how we balance our energy intake. A recent estimate by Sir Stephen O'Rahilly is that "genetics explains most (probably around 65%) of weight variation between individuals" (Speakman and O’Rahilly 2012). The first glimpses on what genes underlie these effects came from mouse breeding experiments in the sixties at Jackson Laboratories in the US. Several monogenic mouse strains were found to have obesity but also comorbidities such as diabetes, and these were aptly named the 1) obese, 2) diabetic, 3) agouti (also has a coat colour phenotype), 4) fat and 5) tubby mice (Naggert et al., 1997). The genes causing these phenotypes were found starting in the nineties and research in this field has exploded since then. Simply, the hormone leptin is produced in adipose tissue in proportion with fat mass and signals via AgRP and POMC neurons to melanocortin neurons that enough energy is present in the periphery. When we fast, leptin (from the greek leptos - thin) levels drop and this gets translated by the melanocortin neurons in the hypothalamus of the brain into hunger. Mice that lack leptin for example (the obese mouse strain) are profoundly hyperphagic because the brain constantly thinks that the animal is starving! Consequently these animals are severely obese with more than half of their body fat being adipose tissue. Administration of leptin corrects the severe obesity, both in mice as well as in people. In a stunning study, Sadaf Farooqi found a girl who gained weight excessively from 4 months of age onwards, and who was constantly hungry. Treatment with leptin cured both (Farooqi et al 1999)!!! While this particular condition is exceedingly rare, the core genes found to be involved in energy homeostasis are conserved between mammals and indeed all vertebrates.

I work on energy homeostasis in the Zebrafish. The zebrafish is an organism whose early development has been extensively studied in the past decades. The genome has been sequenced and labs across the world have contributed to a stunning toolbox to study zebrafish biology. We have established several mutant lines in leptin, leptin receptor and AgRP as well several marker lines in oder to visualize these circuits in early development. While the core genes and mechanisms are conserved between mice, fish and men, key differences exist in the detail. One focus of the lab is to build a roadmap to metabolic control in zebrafish in order to understand which aspects of energy homeostasis are functionally conserved and which differ and how they relate between species.

The recent focus has been purely on leptin biology. The hormone in mice is expressed predominantly in white adipose tissue (Zhang et al 1994) while in fish it is usually not found in adipose tissue at all! We found that overfed zebrafish with leptin or Leptin Receptor mutations do not become obese in contrast to mice with leptin or leptin receptor mutations, however their phenotype lies in glucose regulation. In mice with this mutation, obesity and diabetes are comorbid. This opens up fascinating questions - if leptin does not signal fat levels to the brain, what does? What is the mechanism of glucoregulation by leptin in fish and can  further insight be capitalized on to study why in humans type 2 diabetes is sometimes comorbid with obesity but not always?

The long-term goals of this research are threefold 1) ameliorate abnormal homeostasis patterns (with impact across animal species), 2) gain insight into the evolution of biological diversity and 3) drive insight into growth behavior in farm animals.

Research - active research

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 zebrafish deposit more fat (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).