Genetic inactivation of zinc transporter SLC39A5 improves liver function and hyperglycemia in obesogenic settings
- Regeneron Genetics Center, United States
- Regeneron Pharmaceuticals, United States
- Geisinger Health System, United States
- Department of Clinical Sciences, Sweden
Figures
Figure 1 with 2 supplements
Rare putative LOF (pLOF) variants in SLC39A5 are associated with elevated serum zinc and nominal protection against type II diabetes (T2D).
(A) Serum zinc in carriers of SLC39A5 pLOF variants in the discovery cohort. Controls (Ref; SLC39A5+/+) and heterozygous carriers of pLOF variant alleles in SLC39A5 (Het; SLC39A5+/-). Subject …
Figure 1—figure supplement 1
Graphic abstract.
Heterozygous loss-of-function mutations in SLC39A5 are associated with elevated circulating zinc levels and nominal reduction in type II diabetes risk in humans. Loss of Slc39a5 results in elevated …
Figure 1—figure supplement 2
SLC39A5 putative LOF (pLOF) variants p.Y47*(c.141C>G), p.R311*(c.931C>T), and p.R322*(c.964C>T) encode for non-functional proteins.
HEK293 cell transfected with expression constructs encoding SLC39A5 wild-type (WT), Y47*, R311*, and R322* variants. (A, B) Immunostaining and FACS analysis demonstrating WT SLC39A5 localization to …
Figure 2 with 2 supplements
Loss of Slc39a5 results in elevated circulating and hepatic zinc levels in mice.
Serum zinc (A) and hepatic zinc (B) in Slc39a5+/+, Slc39a5-/-, and Slc39a5+/-mice at 40 wk of age, n=16–18. **p<0.01, ***p<0.001, two-way ANOVA with post hoc Tukey’s test.
Figure 2—figure supplement 1
Generation and characterization of the Slc39a5-/- mice.
(A) Schematic representation of the Slc39a5 null allele. (B) Slc39a5 gene expression in liver and duodenum of Slc39a5-/- mice at 20 wk of age, n=3–6. (C) Immunoblotting analyses demonstrating an …
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Figure 2—figure supplement 1—source data 1
Original files of the full raw uncropped, unedited blots.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Figures with the uncropped blots with the relevant bands clearly labelled.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig2-figsupp1-data2-v1.zip
Figure 2—figure supplement 2
Loss of Slc39a5 does not alter hepatic magnesium, iron, copper, calcium, and cobalt levels in mice challenged with high-fat high fructose diet (HFFD).
Female (A–C) and male (D–F) mice were fed HFFD or NC for 30 wk. (A, D) Densitometric analysis of hepatic AMPK and AKT. (B, E) Hepatic gene expression of Slc39a5 and Mt2. (C, F) Hepatic ion …
Figure 3 with 6 supplements
Loss of Slc39a5 improves glycemic traits in leptin-receptor deficient mice and in mice challenged with high-fat high fructose diet (HFFD).
Female (A-D, I-L; ♀) and Male (E-H, M-P; ♂) mice. (A–H) Slc39a5-/-;Lepr-/- and corresponding control mice. (A, E) Body weight at 34 wk. (B, F) Fasting blood glucose at 34 wk. (C, G) Fasting insulin …
Figure 3—figure supplement 1
Metabolic profiling of female Slc39a5-/-; Lepr-/- mice.
(A) Longitudinal body weight. (B–D) Analyses were done on explanted liver samples collected after 16 hr of fasting at 34 wk of age. (B) Hepatic expression of fatty acid synthase (Fasn) and …
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Figure 3—figure supplement 1—source data 1
Original files of the full raw uncropped, unedited blots.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Figures with the uncropped blots with the relevant bands clearly labelled.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig3-figsupp1-data2-v1.zip
Figure 3—figure supplement 2
Loss of Slc39a5 reduces hepatic fatty acid synthase expression but does not change the insulin profile of male Lepr-/- mice.
(A) Longitudinal body weight. (B–D) Analyses were done on explanted liver samples collected after 16 hr of fasting at 34 wk of age. (B) Hepatic expression of fatty acid synthase (Fasn) and …
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Figure 3—figure supplement 2—source data 1
Original files of the full raw uncropped, unedited blots.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig3-figsupp2-data1-v1.zip
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Figure 3—figure supplement 2—source data 2
Figures with the uncropped blots with the relevant bands clearly labelled.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig3-figsupp2-data2-v1.zip
Figure 3—figure supplement 3
Loss of Slc39a5 reduces hepatic fatty acid synthase expression but does not change insulin profile in female mice challenged with high-fat high fructose diet (HFFD).
(A) Longitudinal body weight during dietary intervention. (B–D) Analyses were done on explanted liver samples collected after 16 hr of fasting in mice fed HFFD or NC for 30 wk. (B) Hepatic …
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Figure 3—figure supplement 3—source data 1
Original files of the full raw uncropped, unedited blots.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig3-figsupp3-data1-v1.zip
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Figure 3—figure supplement 3—source data 2
Figures with the uncropped blots with the relevant bands clearly labelled.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig3-figsupp3-data2-v1.zip
Figure 3—figure supplement 4
Loss of Slc39a5 reduces hepatic fatty acid synthase expression but does not change insulin profile in male mice challenged with high-fat high fructose diet (HFFD).
(A) Longitudinal body weight during dietary intervention. (B–D) Analyses were done on explanted liver samples collected after 16 hr of fasting in mice fed HFFD or NC for 30 wk. (B) Hepatic …
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Figure 3—figure supplement 4—source data 1
Original files of the full raw uncropped, unedited blots.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig3-figsupp4-data1-v1.zip
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Figure 3—figure supplement 4—source data 2
Figures with the uncropped blots with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig3-figsupp4-data2-v1.zip
Figure 3—figure supplement 5
Loss of Slc39a5 improves glycemic traits in Lepr-/- mice and in mice challenged with high-fat high fructose diet (HFFD).
Female (A-D, I-L; ♀) and Male (E-H, M-P; ♂) mice. (A–H) Slc39a5-/-; Lepr-/- and corresponding control mice. (A–B, E–F) Oral glucose tolerance test (GTT) after 16 hr of fasting, at 20 wk. (C–D, G–H) …
Figure 3—figure supplement 6
Additional data of glucose-stimulated insulin secretion, serum hepatic beta-hydroxybutyrate (BHOB), and pancreas histology in mouse models.
(A–H) Oral glucose tolerance test (GTT) was performed in Slc39a5-/- female (A–D) and male (E–H) mice after 16- hr of fasting, at 15 wk. Glucose (A–B, E–F) and insulin (C–D, G–H) levels were measured …
Figure 4 with 1 supplement
Loss of Slc39a5 improves liver function and steatosis in leptin-receptor deficient female mice and in female mice challenged with high-fat high fructose diet (HFFD).
Slc39a5-/-;Lepr-/- and corresponding control mice (A–F) were sacrificed after 16 hr fasting at 34 wk of age. (G–L) Slc39a5-/- and Slc39a5+/+ mice were fed HFFD or NC for 30 wk and sacrificed after …
Figure 4—figure supplement 1
Loss of Slc39a5 improves liver function and steatosis in Lepr-/- male mice and reduces hepatic triglyceride in male mice challenged with high-fat high fructose diet (HFFD).
Slc39a5-/-; Lepr-/- and corresponding control mice (A–F) were sacrificed after 16 hr fasting at 34 wk of age. (G–L) Slc39a5-/- and corresponding control mice were fed HFFD or NC for 30 wk and …
Figure 5 with 4 supplements
Loss of Slc39a5 results in elevated hepatic zinc and activation of hepatic AMPK signaling in leptin-receptor deficient female mice and female mice challenged with high-fat high fructose diet (HFFD).
Analyses were done on explanted liver samples collected after 16 hr of fasting at an endpoint in Lepr-/- (A–C) and HFFD mice (D–F). (A, D) Immunoblot analysis of hepatic AMPK and AKT activation. …
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Figure 5—source data 1
Original files of the full raw uncropped, unedited blots.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig5-data1-v1.zip
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Figure 5—source data 2
Figures with the uncropped blots with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig5-data2-v1.zip
Figure 5—figure supplement 1
Loss of Slc39a5 results in elevated hepatic zinc and activation of hepatic AMPK signaling in congenital and diet-induced obesity models.
Analyses were done on explanted liver samples collected from male mice after 16 hr of fasting at an endpoint of congenital (A–C) and diet-induced obesity (D–F). (A, D) Immunoblot analysis of hepatic …
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Figure 5—figure supplement 1—source data 1
Original files of the full raw uncropped, unedited blots.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Figures with the uncropped blots with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig5-figsupp1-data2-v1.zip
Figure 5—figure supplement 2
Loss of Slc39a5 does not alter hepatic magnesium, iron, copper, calcium, and cobalt levels in Lepr-/- mice.
Female (A–C) and male (D–F) mice were examined at 34 wk of age. (A, D) Densitometric analysis of hepatic AMPK and AKT signaling. (B, E) Hepatic expression of Slc39a5 and Mt2. (C, F) Hepatic ion …
Figure 5—figure supplement 3
Zinc activates AMPK and AKT signaling in a time-dependent and dose-dependent manner.
(A) No differences in cell viability were observed in human primary hepatocytes (after 4 hr treatment) across different experimental groups. (B) Time-resolved (0–4 hr) immunoblotting analyses of …
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Figure 5—figure supplement 3—source data 1
Original files of the full raw uncropped, unedited blots.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig5-figsupp3-data1-v1.zip
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Figure 5—figure supplement 3—source data 2
Figures with the uncropped blots with the relevant bands clearly labeled.
- https://cdn.elifesciences.org/articles/90419/elife-90419-fig5-figsupp3-data2-v1.zip
Figure 5—figure supplement 4
Elevated hepatic zinc results in reduced protein phosphatase activity.
Analyses were done on explanted liver samples collected after 16 hr fast at the endpoint of congenital obesity (A–B) and diet-induced obesity (C–D) challenges. Female (A, C) and Male (B, D) mice. (A–…
Figure 6 with 2 supplements
Loss of Slc39a5 improves hepatic inflammation and fibrosis in female mice challenged with diet-induced non-alcoholic steatohepatitis (NASH).
Slc39a5-/- and Slc39a5+/+ mice were placed on a NASH-inducing diet or NC for 40 wk and sacrificed after 16 hr of fasting. (A, B) NASH Slc39a5-/- mice display reduced serum ALT and AST levels. (C–E) …
Figure 6—figure supplement 1
Loss of Slc39a5 reduces hepatic inflammation and fibrosis in male mice challenged with diet-induced non-alcoholic steatohepatitis (NASH).
Mice were fed NASH diet or NC for 40 wk and sacrificed after 16 hr fasting. (A–B) Loss of Slc39a5 reduces serum ALT and AST levels (biomarkers of liver damage). (C–E) Histology scores for steatosis, …
Figure 6—figure supplement 2
Loss of Slc39a5 improves liver function in mice challenged with diet-induced non-alcoholic steatohepatitis (NASH).
Female (A–E) and Male (F–J) mice. (A, F) Body weight. (B, G) Fasting blood glucose. (C, H) Serum zinc. (D, I) Hepatic zinc. (E, J) Total hepatic superoxide dismutase (SOD) activity. n=6–10 (NC) and …
Additional files
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Supplementary file 1
Serum zinc and insulin profile assessment in the serum call back study.
Serum zinc levels in SLC39A5 heterozygous loss of function carriers are elevated by 12% as compared to age, sex, and BMI-matched reference controls. Analyses of insulin production (insulin/c-peptide ratio), insulin clearance (proinsulin/insulin), and blood glucose in these samples demonstrated no differences based on genotype. Data is represented in a graphical format in Figure 1.
- https://cdn.elifesciences.org/articles/90419/elife-90419-supp1-v1.xlsx
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Supplementary file 2
Tissue zinc content in humans and mouse.
Human data adapted from Jackson et al., 1982 *p<0.05; **p<0.01, ***p<0.001, not significant (n.s.), unpaired t-test. Values represent mean ± SD.
- https://cdn.elifesciences.org/articles/90419/elife-90419-supp2-v1.xlsx
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Supplementary file 3
No differences in the serum chemistry profile of Slc39a5+/+ and Slc39a5-/- mice.
Serum chemistry analysis in adult mice (40 wk of age, both sexes) demonstrated no differences in pancreatic amylase, renal function parameters (blood urea nitrogen, creatinine, total protein, and uric acid), and electrolytes (chloride, potassium, and sodium) or liver enzymes (alanine aminotransferase; ALT and aspartate aminotransferase; AST).
- https://cdn.elifesciences.org/articles/90419/elife-90419-supp3-v1.xlsx
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Supplementary file 4
Summary statistics for the congenital obesity model.
Loss of Slc39a5 improves glycemic traits and liver function in leptin-receptor (Lepr) deficient mice. Loss of Slc39a5 does not change insulin production (proinsulin/insulin), or insulin clearance (insulin/c-peptide ratio).
- https://cdn.elifesciences.org/articles/90419/elife-90419-supp4-v1.xlsx
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Supplementary file 5
Summary statistics for the diet-induced obesity model.
Loss of Slc39a5 improves glycemic traits and liver function in mice upon a high-fat high fructose diet (HFFD) dietary challenge. Moreover, loss of Slc39a5 does not change insulin production (proinsulin/insulin), or insulin clearance (insulin/c-peptide ratio).
- https://cdn.elifesciences.org/articles/90419/elife-90419-supp5-v1.xlsx
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Supplementary file 6
Summary statistics for the diet-induced non-alcoholic steatohepatitis (NASH) model.
Loss of Slc39a5 improves hepatic inflammation and fibrosis in both female and male mice challenged with diet-induced NASH.
- https://cdn.elifesciences.org/articles/90419/elife-90419-supp6-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/90419/elife-90419-mdarchecklist1-v1.docx
