Accumulation of fat in liver of leptin-deficient mice is influenced by FTO.

<p>a) Liver weight in relation to body length (n = 13, 14, 16, 14). *indicate significant p-values between <i>Lep^ob/ob;Fto^+/+</i>and <i>Lep^ob/ob;Fto^−/−</i>. b) Haematoxylin and eosin staining on paraffin sections (upper row) and oil red O staining on cryo sections (lower row) for all 4 genotypes. Scale = 100 µm. Livers were used from female mice at the age of 30 weeks. All data are presented as mean. Error bars indicate the SEM.</p

Detailed analysis of adipose tissue.

<p>All data are collected from 30 weeks old mice. *indicate significant p-values between <i>Lep^ob/ob;Fto^+/+</i> and <i>Lep^ob/ob; Fto^−/−</i>. a) Weights of different fat pads from female mice (n = 13, 16, 18, 16). b) Area size of epigonadal fat cells from female mice (n = 4, 4, 8, 7). c+d) Expression analysis for different marker genes of epigonadal adipose tissue (n = 6, 4, 5, 5, 5). Following p-values were calculated: between <i>Lep^ob/ob;Fto^+/−</i> and <i>Lep^ob/ob; Fto^−/−</i>: PPARγ2: p = 0,08, Adiponectin: p = 0,21, TNFα:p = 0,06, IL-6:p = 0,03. Data are presented as mean. Error bars indicate the SEM.</p

FTO contributes to the development of hyperglycaemia.

<p>All data are elevated from female mice. *indicate significant p-values between <i>Lep^ob/ob;Fto^+/+</i>and <i>Lep^ob/ob;Fto^−/−</i>. a) Fasted glucose levels in mice at the age of 6 weeks (n = 13, 10, 12, 8) b) Non-fasted glucose level at the age of 7 weeks (n = 12, 9, 12, 9). c) Glucose tolerance test (GTT) at the age of 6 weeks (n = 13, 10, 12, 8). d) Insulin tolerance test (ITT) at the age of 7 weeks (n = 12, 9, 12, 9). Glucose values are presented relative to initial glucose levels. e) Fasted glucose levels in mice at the age of 14 weeks (n = 11, 7, 9, 9). f) Non-fasted glucose level at the age of 15 weeks (n = 11, 8, 14, 12). g) GTT at the age of 14 weeks (n = 11, 7, 9, 9). h) ITT at the age of 15 weeks (n = 11, 8, 14, 12). Glucose values are presented relative to initial glucose levels. i) Plasma glucose levels of 30 weeks old non fasted mice (n = 8, 5, 5, 6). j) Plasma insulin levels of non-fasted mice at the age of 30 weeks (n = 3, 3, 7, 3). All data are presented as mean. Error bars indicate the SEM.</p

FTO contributes to gain of body weight in <i>Lep^ob/ob</i> mice.

<p>All data are collected from female mice. a) Development of body weight from 3 to 16 weeks of age. n(<i>Lep^+/+; Fto^+/+</i>) = 15–20, n(<i>Lep^ob/ob;Fto^+/+</i>) = 11–13, n(<i>Lep^ob/ob;Fto^+/−</i>) = 15–19, n(<i>Lep^ob/ob;Fto^−/−</i>) = 9–12, n(<i>Lep^+/+;Fto^−/−</i>) = 4–6. Asterisks (*) indicate significant p-values between <i>Lep^ob/ob;Fto^−/−</i> and <i>Lep^ob/ob;Fto^+/+</i>. Further significant p-values were found: <i>Lep^+/+;Fto^+/+</i>–<i>Lep^ob/ob;Fto^−/−</i>: p≤0,05 (weeks 3–8+11–16); <i>Lep^+/+;Fto^+/+</i>–<i>Lep^ob/ob;Fto^+/+</i>: p≤0,05 (weeks 4–16); <i>Lep^+/+;Fto^+/+</i>–<i>Lep^ob/ob;Fto^+/−</i>: p≤0,05 (weeks 4–16); <i>Lep^ob/ob;Fto^−/−</i>–<i>Lep^ob/ob;Fto^+/−</i>: p≤0,05 (weeks 3–16); <i>Lep^ob/ob;Fto^+/+</i>–<i>Lep^ob/ob;Fto^+/−</i>: p≤0,05 (weeks 4–14);; <i>Lep^+/+;Fto^+/+</i>–<i>Lep^+/+;Fto^−/−</i>: p≤0,05 (weeks 3–16). b–e) *indicate significant p-values to <i>Lep^ob/ob;Fto^+/+</i>. b) Body length at the age of 30 weeks (n = 14, 15, 17, 15, 10). c) Body weight in relation to body length at the age of 9 (n = 6, 6, 7, 5, 5), 16 weeks (n = 4, 6, 7, 5, 4) and 30 weeks (n = 14, 15, 17, 15, 10) of age. d) Lean mass and fat mass at the age of 8 weeks (n = 7, 8, 4, 4, 3). e) Lean mass and fat mass at the age of 16 weeks (n = 12, 7, 9, 4, 3). All data are presented as mean. Error bars indicate the SEM.</p

Increase of pancreatic islet size in leptin-deficient mice is depending on FTO.

<p>a) Haematoxylin and eosin staining on paraffin sections of the pancreas for all 4 genotypes. b) Immunofluorescence staining for insulin (red) and glucagon (green) on pancreas sections. Nuclei are stained with DAPI. Scale = 100 µm. c) Area of beta cells in relation to full pancreas section area. d) Number of islets of Langerhans which were found on sections categorized into 3 groups of size. Pancreas were taken from 30 weeks old female mice (n = 5, 6, 7, 5). All data are presented as mean. Error bars indicate the SEM.</p

Effects of the small molecule combination.

<p>A. ASH cells transduced with <i>Ad-PNM</i>, without and with the DNB combination. PDX1 is immunostained green and insulin red. Scale bar 100µm. B. Percentage of PDX1-positive cells that are also insulin-positive. Comparisons are by one way ANOVA with Games-Howell post hoc test, * indicates significant enhancement. Errors are standard errors, n=6.</p

Insulin-positive cells generated from different cell types.

<div><p>A,B,C Expression of PDX1, NGN3, MAFA in ASH cells. The proteins are shown in green, DAPI in blue.</p> <p>D. Histogram of the percentage PDX1-positive cells which are insulin-positive in different cell types. </p> <p>E,E’ Rat hepatocytes, some PDX1-positive cells are insulin-positive. </p> <p>F,F’ Rat hepatocytes, insulin-positive cells are also C-peptide-positive. </p> <p>G Mouse ASH cells, some PDX1-positive cells are insulin-positive. </p> <p>H. Mouse ASH cells, showing cytoplasmic location of insulin. Inset, high magnification showing insulin-containing granules.</p> <p>I,I’ Mouse ASH cells showing co-expression of C-peptide with insulin. </p> <p>J Rat AR42j-B13 cells showing a high proportion of insulin-positive cells. </p> <p>K,K’ Rat AR42j-B13 cells showing co-expression of C-peptide with insulin. </p> <p>L. Rat AR42j-B13 showing insulin-containing granules.</p> <p>Scale bars 100µm, except H inset and L which are 25 µm. Blue color is DAPI throughout.</p></div

EdU labeling index in five cell types.

<p>Those which generated insulin-immunopositive cells showed a reduction in EdU label. Errors are standard errors, n=3.</p

Expression of various beta cell genes provoked by transduction with <i>Ad-PNM</i>.

<p>A-H. Qualitative RT-PCRs showing the effect of <i>Ad-PNM</i> on the expression of a panel of beta cell genes. 30 cycles were used unless otherwise shown. The misaligned bands in the first two lanes of mouse fibroblast <i>Mnx1</i> are primer dimers, not RNA.</p

Schematic representation of the possible pathways of differentiation of B13 cells into hepatocytes and ductal cells.

<p>Diagram illustrating the potential relationship between pancreatic acinar cells, ductal cells and hepatocytes.</p