Hyperphagia results in hyperplastic and hypertrophic adipocytes in middle-aged fish.

<p>A-H: H&E staining of longitudinal sections of male (A,B,E,F) and female (C,D,G,H) HF-LD (A,C,E,G) and LF-HD (B,D,F,H) fish, 6.5 months of age, dorsoventral level 2 (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120776#pone.0120776.s004" target="_blank">S4A Fig</a>.); I-J: Average total numbers of subcutaneous adipocytes per level (I) and sizes (J) of subcutaneous adipocytes of male and female LF-HD and HF-LD fish (body length 28.8 +/- 0.9 mm (male HF-LD), 21.2 +/- 1.5 mm (male LF-HD), 31.1 +/- 1.4 mm (female HF-LD) and 20.8 +/- 1.4 mm (female LF-HD)) from 5 corresponding longitudinal levels along the dorsoventral axis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120776#pone.0120776.s004" target="_blank">S4C,D Fig</a>.); columns with different superscript letters are significantly different (p<0.05) according to ANOVA followed by the Least Significant Difference (Bonferroni’s) test, n = 40 (2 fish per condition, 5 levels per fish, 2 sections per level and left and right sides per section). Similar experiments were obtained in an additional, independent experiment. Abbreviations: im, intermuscular adipocytes; s, scale; sc, subcutaneous adipocytes; vb, vertebral body.</p

Aged (18 months old) male HF-LD and LF-HD fish display decreased adipocyte sizes but unaltered adipocyte numbers compared to middle-aged fish.

<p>A-H: H&E staining on longitudinal sections of male LF-HD (E-H) and HF-LD (A-D) fish showing visceral (A,E), subcutaneous (D,H) and intermuscular (B,C,F,G) adipocytes at dorsoventral levels 2 (A,B,D,E,F,H) or level 4 (C,G) (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120776#pone.0120776.s005" target="_blank">S5A Fig</a>.); I,J: Average total numbers of subcutaneous adipocytes per level (I) and sizes (J) of subcutaneous adipocytes of male LF-HD and HF-LD fish (body length 32.3 +/- 1.0 mm (male HF-LD), 22.5 +/- 1.0 mm (male LF-HD)) from 5 corresponding longitudinal levels along the dorsoventral axis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120776#pone.0120776.s005" target="_blank">S5C,D Fig</a>.); *** indicates significant differences (p<0.001) according to the Student`s T test, n = 40 (2 fish per condition, 5 levels per fish, 2 sections per level and left and right per section). Similar results were obtained in an additional, independent experiment. Abbreviations: im, intermuscular adipocytes; s, scale; sc, subcutaneous adipocytes; vb, vertebral body; vc, visceral adipocytes.</p

Hyperphagia leads to increased ovarian sizes and enhanced oocyte growth rates, while not affecting final oocyte sizes.

<p>A-B: Weight of ovaries of female LF-HD, NF-ND and HF-HD in mg (A) and in % total body weight (B) at different ages; at an age of 2 months, numbers could only be supplied for HF-LD females, since NF-ND and LF-HD fish of this age were not sexually mature as yet; columns with same superscript letter are not significantly different (p>0.05) according to ANOVA followed by the Least Significant Difference (Bonferroni’s) test, n ≥ 5. C: Sizes of fully grown oocytes and total number of oocytes of female LF-HD and HF-LD fish were not significantly (n.s.) different according to the Student`s T test, n = 10 (10 biggest oocytes of all levels) or n = 3 (3 females). D: Relative numbers of oocytes per maturation stage of HF-LD and LF-HD females (in % of total oocyte numbers) in all of the four analyzed dorsoventral levels (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120776#pone.0120776.s006" target="_blank">S6A Fig</a>.); ** indicates significant differences (p<0.01), n.s. means not significant according to the Student`s T test, n = 3. E-F: H&E staining of longitudinal sections through ovaries of HF-LD (E) and LF-HD female (F), 8.5 months of age, dorsoventral level 2. I: primary oocyte, stage 1–2 (previtellogenic); II: primary oocyte, stage 3–5 (previtellogenic); III: primary oocyte, stage 6 (vitellogenic); IV: secondary oocyte (meiotic arrest), stage 7; staging was done as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120776#pone.0120776.ref071" target="_blank">71</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120776#pone.0120776.ref072" target="_blank">72</a>]. Similar results were obtained in second, independent experiments.</p

Hyperphagia results in increased linear growth and earlier scale formation

<p>A-D: Alizarin red staining of age-matched (28 dpf) juvenile fish (A,C) and corresponding magnification of the flank (B,D) of fish raised with high amounts of food at low density (A,B; HF-LD) and low amounts of food at high density (C,D; LF-HD). Body lengths of the shown individuals are indicated. Regions magnified in (B,D) are boxed in (A,C). E: Graph summarizing scale mineralization status (number of rows of mineralized scales) of analyzed fish at different ages. HF-LD fish started to display 7 rows of mineralized from an age of 27 days onwards, while it took LF-HD fish at least 34 days to reach this stage. F: Body length growth curves of sibling fish as in (A-E) shown as mean +/- standard deviation of 10 analyzed fish per condition and time point. LF-HD fish are significantly shorter than HF-LD fish. Similar results were obtained in a second, independent experiment. Abbreviations: ha, hemal arch; dpf, days post-fertilization; na, neural arch; s, scale; vb, vertebral body.</p

Caloric intake has differential effects on the timing of scale formation, somatic and ovarian growth and fat incorporation.

<p>A-B: Alizarin red staining of size-matched juvenile HF-LD (A) and LF-HD (B); body lengths and ages of the shown individuals are indicated. The flank of the LF-HD fish has several rows of mineralized scales, whereas no scales are visible in the corresponding region of the HF-LD fish. (C) Plot of rows of mineralized scales vs body length. (D,E) Comparison of numbers (D) and sizes (E) of subcutaneous adipocyte sizes (E) of 18 months old LF-HD males (white columns) with age-matched HF-LD male siblings (black column) and size-matched HF-LD males (striped columns; age: 2 months). Standard lengths of investigated fish were: HD-LD, 18 months: 32.3 +/- 1.0 mm; HF-LD, 2 months: 23.0 +/- 0,8 mm; LF-HD, 18 months; 22.5 +/- 1.0 mm. Left sides of each panel show average numbers from 5 corresponding longitudinal levels along the entire dorsoventral axis (for averages of single levels, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120776#pone.0120776.s007" target="_blank">S7 Fig</a>.), right sides show numbers for level 5 only (ventral). Columns with different superscript letter are significantly different (p<0.05) according to ANOVA followed by the Least Significant Difference (Bonferroni’s) test; n = 40 (2 fish per condition, 5 levels per fish, 2 sections per level and left and right side per section). F-J: Plots of BMI vs length of males (F); somatic BMI vs. length of females (G); absolute ovary weight vs. length (including 2 months HF females) (H); relative ovary weight vs. length (I); relative ovarian weight vs. somatic BMI (J). Crucial parameter intervals in (C, F-J) are boxed in red or blue, intervals only occupied by HF-LD fish are highlighted in blue, intervals only occupied by LF-HD fish in red. In (G-J), HF-LD females in regions boxed in red were 2 months of age, but already sexually mature. K: Schemes showing differential energy allocations to scale formation / postembryonic development, growth, reproduction and fat storage in juvenile, small and large adult fish. Abbreviations: ha, hemal arch; na, neural arch; s, scale; vb, vertebral body.</p

Long-term obesogenic diet leads to metabolic phenotypes which are not exacerbated by catch-up growth in zebrafish

Obesity is a world wide problem and evidence suggests, that early lifetime undernourishment of caloric restirction predispose an organism for obesity and metabolic syndrome. We have raised two cohorts of zebrafish in an obesogenic environment (DIO) and compared several metabolic markers with fish raised under caloric restriction (CR) or fish shifted from CR to DIO at different periods in their life. We have looked morphologically at standard length and weight and found that fish on DIO grow faster in both axes. Fish shifted from CR to DIO show catch-up growth and not compensatory growth when shifted at one month, 3 months or 9 months of age. We have further characterized central agrp expression and hyperphagia, adipose tissue by histology as well as uCT imaging, hepatic histology, metabolic rate mitochondrial function as well as feeding induced glucose levels. We find that fish in an obesogenic environment develop markers of obesity which are not exacerbated by ealry lifetime food restriction

Micro CT scans for zebrafish on an obesogenic diet (DIO), on caloric restriction (CR) or undergoing catch up growth after 1, 3 or 9 months of CR (CG1, CG3, CG9)

For µCT imaging, adult zebrafish were fixed and decalcified in Bouin's solution at room temperature for 7 days, stored in PBS and imaged using a micro-computed tomography (µCT) device (SkyScan1272, Bruker BioSpin GmbH, Ettlingen, Germany). Zebrafish were placed individually in 1.5ml Eppendorf tubes using and an ultra-focus scan over the whole body was performed in a full-rotation in step-and-shoot mode. 322 projections (1008x672 pixels, 4x4 binning) were acquired per subscan with an x-ray tube voltage of 60 kV, power 0.166 mA, aluminum filter 0.25 mm,exposure time of 363 ms, 6 averages and a object-source distance of 86 mm. All CT images were reconstructed at an isotropic voxel size of 18 µm using a Feldkamp type algorithm (filtered back-projection). Fat-containing regions were appear hypo intense in µCT data and were segmented using Imalytics Preclinical (Gremse-IT GmbH, Aachen, Germany (Gremse et al., 2016; doi:10.7150/thno.13624). The volumetric fat percentage was calculated as the ratio of subcutaneous adipose tissue (SAT) or visceral adipose tissue (VAT) fat volume compared to the entire volume of the body cavity anterior of the anal fin and expressed per skeletal segment

Long-term obesogenic diet leads to metabolic phenotypes which are not exacerbated by catch-up growth in zebrafish.

Obesity and metabolic syndrome are of increasing global concern. In order to understand the basic biology and etiology of obesity, research has turned to animals across the vertebrate spectrum including zebrafish. Here, we carefully characterize zebrafish in a long-term obesogenic environment as well as zebrafish that went through early lifetime caloric restriction. We found that long-term obesity in zebrafish leads to metabolic endpoints comparable to mammals including increased adiposity, weight, hepatic steatosis and hepatic lesions but not signs of glucose dysregulation or differences in metabolic rate or mitochondrial function. Malnutrition in early life has been linked to an increased likelihood to develop and an exacerbation of metabolic syndrome, however fish that were calorically restricted from five days after fertilization until three to nine months of age did not show signs of an exacerbated phenotype. In contrast, the groups that were shifted later in life from caloric restriction to the obesogenic environment did not completely catch up to the long-term obesity group by the end of our experiment. This dataset provides insight into a slowly exacerbating time-course of obesity phenotypes

Diet-Induced Growth Is Regulated via Acquired Leptin Resistance and Engages a Pomc-Somatostatin-Growth Hormone Circuit

Summary: Anorexigenic pro-opiomelanocortin (Pomc)/alpha-melanocyte stimulating hormone (αMSH) neurons of the hypothalamic melanocortin system function as key regulators of energy homeostasis, also controlling somatic growth across different species. However, the mechanisms of melanocortin-dependent growth control still remain ill-defined. Here, we reveal a thus-far-unrecognized structural and functional connection between Pomc neurons and the somatotropic hypothalamo-pituitary axis. Excessive feeding of larval zebrafish causes leptin resistance and reduced levels of the hypothalamic satiety mediator pomca. In turn, this leads to reduced activation of hypophysiotropic somatostatin (Sst)-neurons that express the melanocortin receptor Mc4r, elevated growth hormone (GH) expression in the pituitary, and enhanced somatic growth. Mc4r expression and αMSH responsiveness are conserved in Sst-expressing hypothalamic neurons of mice. Thus, acquired leptin resistance and attenuation of pomca transcription in response to excessive caloric intake may represent an ancient mechanism to promote somatic growth when food resources are plentiful. : The melanocortin system controls energy homeostasis and somatic growth, but the underlying mechanisms are elusive. Löhr et al. identify a functional neural circuit in which Pomc neurons stimulate hypothalamic somatostatin neurons, thereby inhibiting hypophyseal growth hormone production. Excessive feeding and acquired leptin resistance attenuate this pathway, allowing faster somatic growth when food resources are rich. Keywords: Pomc neuron, somatostatin neuron, somatic growth, growth hormone, melanocortin system, high-fat diet, obesity, leptin resistance, zebrafish, mous