miR-375-null mice develop diabetes. (A) Random-fed blood glucose levels in 375KO (filled squares) and wild-type littermate control (open circles) male mice. (B and C) Plasma insulin and glucagon levels in 10-week-old 375KO mice (black bars) and wild-type (gray bars) male mice. (D) Intraperitoneal glucose tolerance test administered to 10-week-old mice. (E) Plasma insulin levels during i.p. glucose tolerance test. (F) Insulin secretion of isolated islets in response to indicated glucose concentrations. (G) Insulin tolerance test of 375KO and wild-type littermates (n = 5).

Regulation of gene expression and identification of growth target genes in 375KO islets. (A) Quantification of percentage of Ki-67-positive nuclei within insulin-positive cells of 375KO (black bars) and wild-type (gray bars) male mice. (B) Analysis of gene expression of putative miR-375 targets by real-time PCR in mutant and wild-type pancreatic islets. n = 5 animals per genotype. (C) Western blot analysis of protein lysates from pancreatic islets isolated from 375KO and wild-type (WT) male mice (100 islets per lane). Quantitative measurements made from densitometry are expressed as a ratio of mean values of 375KO to wild-type mice. (D) Increase in intracellular concentration of miR-375 decreases luciferase activity in HEK293 cells transfected with reporter constructs containing either full-length or partial 3′UTR sequence of putative miR-375 target genes (n = 6). Values relative to luciferase activity from cells transfected with a scrambled control are shown. Data are presented as means ± SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Impaired β-cell proliferation in miR-375/ob double-knockout mice. (A) Relative miR-375 expression in lep^ob/lep^ob (ob/ob) mice and wild-type (WT) controls measured by real-time PCR and normalized to U6 (N1) or miR-107 (N2) expression levels. (B) Representative 8-μm sections of pancreas from 10-week-old 375/ob (miR-375^−/− leptin^−/−) and ob/ob mice visualized by immunofluorescence after staining with insulin (green) and glucagon (red). (Bar, 50 μm.) (C) β-Cell mass in 10-week-old WT (gray bar), 375KO (black bar), ob/ob (dark gray bar), and 375/ob (open bar) mice is quantified and reported as mean ± SE. (D–F) Quantification of β-cell number (insulin-positive cells), total endocrine cell number (insulin, glucagon, and somatostatin-positive cells) and α-cell number (glucagon-positive cells) per total pancreatic area in wild-type (gray bars), 375KO (black bars), ob/ob (dark gray bars), and 375/ob (open bars) 10-week-old mice. (G) Ratio of α-cell number to islet cell number in wild-type (gray bar), 375KO (black bar), ob/ob (dark gray bar), and 375/ob (open bar) 10-week-old mice. (H) Quantification of percentage of Ki-67 insulin-positive nuclei within insulin-positive cells of 10-week-old 375/ob (black bar) and ob/ob (gray bar) mice. n > 30 for each genotype. (I) Random-fed blood glucose levels in 375/ob (open squares), ob/ob (filled circles), 375KO (filled squares), and wild-type littermate control (WT) (open circles) mice. (J) Plasma insulin levels in random-fed, 10-week-old 375/ob (black bar) and ob/ob (gray bar) mice. (K) Hepatic glucose production measured after sodium [2-¹⁴C]pyruvate was administered by i.p. injection into random-fed, 10-week-old 375/ob (black bar) and ob/ob (gray bar) mice. Data are presented as means ± SE. n = 4–6 animals per genotype unless otherwise noted. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Decreased β-cell mass in 375KO pancreatic islets. (A) β-Cell mass in wild-type (gray bars) and 375KO (black bars) mice is quantified and reported as mean ± SE. (B–E) Quantification of endocrine cell number per total pancreatic area, β-cell number (B), total endocrine cell number per total pancreatic area (insulin, glucagon, and somatostatin-positive cells) (C), α-cell number (D), and δ-cell number (E) in 375KO (black bar) and wild-type (gray bar) male mice. (F) Representative sections of pancreas from 10-week-old 375KO and wild-type male mice visualized by immunofluorescence after staining with anti-insulin (green) and anti-glucagon (red) antibodies. (Bar, 50 μm.) (G) Glucagon secretion measured from islets isolated from 10-week-old male 375KO (black bars) and wild-type (gray bars) mice cultured overnight and incubated in fresh medium containing the indicated glucose concentrations. (H) Intraperitoneal pyruvate tolerance test was performed on random-fed 6-week-old male mice by administering a dose of sodium pyruvate (in saline) at 2 g/kg body weight. (I) [2-¹⁴C]Pyruvate was administered by i.p. injection into random-fed 6-week-old 375KO and wild-type (WT) mice and blood was drawn after 30 min and deproteinized, and labeled glucose in supernatant was recovered and radioactivity was measured. (J) Quantification of PEPCK and G6Pase mRNA expression by real-time PCR in liver from random-fed, 10-week-old 375KO (375KO) and wild-type (WT) mice. n = 5–12 animals per genotype unless otherwise noted. Data are presented as means ± SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001.