Journal: iScience

Article Title: Zhx2 maintains islet β-cell mass and function by transcriptionally regulating Pax6.

doi: 10.1016/j.isci.2023.106871

Figure Lengend Snippet: Figure 5. Zhx2 maintains islet b-cell function depending on Pax6 MIN6 cells were transfected with si-NC or si-Zhx2 for 12 h and then infected with Ad-GFP or Ad-mPax6 for 48 h. (A) The percentage of MIN6 cell proliferation versus control was measured by flow cytometry. Flow cytometry histograms showing the level of EdU in MIN6 cell lines (n = 3). (B) MIN6 cells were treated with 2.8 or 25 mM glucose. Insulin levels in the culture supernatants were determined by ELISA (n = 3). (C) qRT-PCR was performed to detect the indicated gene expression. mRNA levels were normalized to b-actin mRNA (n = 3). (D) Islets were infected with Ad-GFP or Ad-mPax6 for 48 h, and qRT-PCR was performed to detect the indicated gene expression. mRNA levels were normalized to b-actin mRNA (n = 3). (E) MIN6 cells infected with GV287 (Vector) or GV287-Zhx2 lentiviruses and transfected with si-NC or si-Pax6 for 72 h. Protein levels of these genes were measured by Western blotting. (F) MIN6 cells were co-transfected with si-NC or si-Pax6 and the pc3.0 or pcZhx2 plasmid for 72 h. The protein levels of these genes were measured by Western blotting and analyzed by ImageJ assay. Data are presented as the mean G SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns (no significant difference) by Student’s t test.

Article Snippet: The insulin levels were measured by a mouse insulin ELISA kit (Elabscience, China) according to the manufacturer’s instructions.

Techniques: Cell Function Assay, Transfection, Infection, Control, Cytometry, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Gene Expression, Plasmid Preparation, Western Blot

Journal: Nature Metabolism

Article Title: LONP1 regulation of mitochondrial protein folding provides insight into beta cell failure in type 2 diabetes

doi: 10.1038/s42255-025-01333-7

Figure Lengend Snippet: ( a ) Model of Cre-mediated recombination of the LonP1 locus. ( b ) Ad libitum -fed body weight measured in littermate mice between ages 5–7 weeks. n = 5 Ins1 +/+ ; LonP1 loxP/loxP vs 8 Ins1 Cre/+ ; LonP1 +/+ at 5 weeks; n = 7 Ins1 +/+ ; LonP1 loxP/loxP vs 5 Ins1 Cre/+ ; LonP1 +/+ at 7 weeks. ( c ) Blood glucose concentrations measured during IPGTT in littermate mice at age 5 weeks (left) and age 7 weeks (right). n = 5 Ins1 +/+ ; LonP1 loxP/loxP vs 8 Ins1 Cre/+ ; LonP1 +/+ at 5 weeks; n = 7 Ins1 +/+ ; LonP1 loxP/loxP vs 5 Ins1 Cre/+ ; LonP1 +/+ at 7 weeks. ( d ) Blood glucose concentrations measured during IPGTT related to Fig. . n = 8 male vs 6 female/Ctrl group and 7 male vs 4 female/ β-LonP1 KO group. ( e ) Ad libitum -fed body weight measured in mice between ages 4–10 weeks. n = 10 Ctrl vs 8 β-LonP1 KO at 4 weeks; n = 10 Ctrl vs 8 β-LonP1 KO at 6 weeks; n = 6 Ctrl vs 5 β-LonP1 KO at 10 weeks. ( f ) Blood glucose concentrations (presented as % of baseline glucose) measured during insulin tolerance testing (ITT) of 4-week-old (left) and 6-week-old (right) mice. n = 5 Ctrl vs 3 β-LonP1 KO at 4 weeks; n = 10 Ctrl vs 6 β-LonP1 KO at 6 weeks. ( g ) Glucose-stimulated insulin secretion following static incubations in 2 mM and 16.7 mM glucose, performed in isolated islets of 6-week-old mice (normalized to total insulin content). n = 10 mice/group. ( h ) Pancreatic β cell mass related to Fig. . n = 3 male vs 4 female/Ctrl group and 4 male vs 2 female/ β-LonP1 KO group at 4 weeks; n = 5 male vs 3 female/Ctrl group and 3 male vs 3 female/ β-LonP1 KO group at 6 weeks; n = 3 male vs 3 female/Ctrl group and 4 male vs 1 female/ β-LonP1 KO group at 10 weeks. ( i ) Quantification of cell death ELISA (normalized to total DNA content) measured in isolated islets of 6-week-old mice. n = 7 mice/group. ( j ) Representative immunofluorescence images ( n = 5/group) depicting β cell maturity (left) or dedifferentiation markers (right) from pancreatic sections of 6-week-old mice. A representative image of pancreatic sections of high fat diet-fed of β-Clec16a KO mice as a positive control for ALDH1A3 immunostaining. Scale bars, 50 μm. All data in figure are presented as mean ± SEM. Statistical analysis: 5 G, * *P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparisons test. 5I, *P < 0.05 by unpaired two-tailed Student’s t -test.

Article Snippet: To generate β-cell-specific deletion, floxed models were crossed to Ins1 Cre mice from The Jackson Laboratory (strain no. 026801).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Positive Control, Immunostaining, Two Tailed Test

Journal: Nature Metabolism

Article Title: LONP1 regulation of mitochondrial protein folding provides insight into beta cell failure in type 2 diabetes

doi: 10.1038/s42255-025-01333-7

Figure Lengend Snippet: a , Representative flow cytometry histogram demonstrating cellular ROS (left) and quantification of relative ROS levels (right) in dispersed islets isolated from 6-week-old mice. n = 4 Ctrl versus 3 β- Lonp1 KO. b , Representative immunoblot images (left) and densitometry (right) of phospho-γH2AX in islets isolated from 6-week-old mice. n = 3 mice per group. c , Quantification of relative ROS levels in Min6 β cells 72 h after transfection with siLonp1 or siCtrl and treated with 5 mM NAC or vehicle Ctrl for the final 36 h. n = 7 per group. d , GSIS (left) after static incubation in 2 mM and 16.7 mM glucose, and islet insulin content (right), measured in human islets treated with or without 1 µM CDDO and with or without 5 µM NAC for 24 h. n = 4 independent islet donors per group. e , Quantification of cell death measured in human islets treated with or without 1 µM CDDO and with or without 5 mM NAC for 24 h. n = 4 independent islet donors per group. f , Expression of catalase and LONP1 using immunoblotting in islets isolated from 5–7-week-old mice. Representative of three mice per group. g , Quantification of relative ROS levels determined using flow cytometry in the isolated islets of 5-week-old mice. n = 3 mice per group. h , Blood glucose concentrations measured during IPGTT from 5-week-old Ins1 Cre ( n = 15), mCAT; Ins1 Cre ( n = 6), β- Lonp1 KO ( n = 8) and mCAT; β- Lonp1 KO mice ( n = 13). i , GSIS (left) after static incubation in 2 mM and 16.7 mM glucose, and islet insulin content (right) measured in isolated islets of 5-week-old Ins1 Cre ( n = 11), mCAT; Ins1 Cre ( n = 10), β- Lonp1 KO ( n = 7) and mCAT; β- Lonp1 KO ( n = 6) mice. j , Pancreatic β cell mass measured in 5-week-old Ins1 Cre ( n = 6), mCAT; Ins1 Cre ( n = 4), β- Lonp1 KO ( n = 6) and mCAT; β- Lonp1 KO ( n = 6) mice. k , Quantification of β cell death using TUNEL and insulin immunostaining from pancreatic sections of 5-week-old Ins1 Cre ( n = 6), mCAT; Ins1 Cre ( n = 4), β- Lonp1 KO ( n = 5) and mCAT; β- Lonp1 KO ( n = 6). l , Expression of phospho-γH2AX using immunoblotting. m , Phospho-γH2AX densitometry in islets isolated from 5-week-old mice. n = 3 mice per group. n , Quantification of relative ROS production in the islets of 7-week-old mice. n = 3 mice per group. o , Blood glucose levels measured during IPGTT from 7-week-old Ins1 Cre ( n = 11), mCAT; Ins1 Cre ( n = 4), β- Lonp1 KO ( n = 7) and mCAT; β- Lonp1 KO ( n = 9) mice. p , GSIS (left) after static incubation in 2 mM and 16.7 mM glucose, and islet insulin content (right) measured in the isolated islets of 7-week-old Ins1 Cre ( n = 9), mCAT; Ins1 Cre ( n = 7), β- Lonp1 KO ( n = 7) and mCAT; β- Lonp1 KO ( n = 7) mice. q , β cell mass determined in pancreatic sections of 7-week-old mice. n = 6 mice per group. r , Quantification of TUNEL staining performed in pancreatic sections of 7-week-old mice for β cell apoptosis. n = 6 mice per group. All data are presented as the mean ± s.e.m. a , b , *P < 0.05 and * *P < 0.01 were determined using an unpaired, two-tailed Student’s t -test. c – e , g – k , m – r , *P < 0.05 and * *P < 0.01 were determined using a one-way ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: To generate β-cell-specific deletion, floxed models were crossed to Ins1 Cre mice from The Jackson Laboratory (strain no. 026801).

Techniques: Flow Cytometry, Isolation, Western Blot, Transfection, Incubation, Expressing, TUNEL Assay, Immunostaining, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Reprogramming insulin receptor activation with a de novo agonist to overcome severe insulin resistance

doi: 10.64898/2026.05.04.722722

Figure Lengend Snippet: (A) Structural view of insulin binding at site-1 of the insulin receptor (IR). Asp707 (red) within the αCT motif (blue) of the IR is a key residue mediating site-1 insulin interaction. (B) Representative images of WT, D707A/+, and D707A/D707A littermates at embryonic day 18.5 and at birth. (C) Genotype distribution of WT, D707A/+, and D707A/D707A mice at different ages. (D) Hematoxylin and Eosin (H&E) staining and periodic acid–Schiff (PAS) staining (magenta) of livers from WT and D707A/D707A newborn mice. (E) Breeding strategy used to generate conditional D707A/– mice. (F) Experimental scheme. Male and female mice (2–3 months old) were injected with tamoxifen for five consecutive days to delete the floxed WT IR allele and generate D707A/– mice. Data from male mice are shown in (G-L) and data from female mice are shown in . (G) Ad libitum blood glucose levels. D707A/F, n = 10; D707A/–, n = 12; –/–, n = 15. Data are presented as mean ± SEM; one-way ANOVA. (H) Fasting insulin levels. D707A/F, n = 9; D707A/–, n = 13; –/–, n = 7. Data are presented as mean ± SEM; one-way ANOVA. (I) Glucose tolerance test (GTT). Data are presented as mean ± SEM; two-way ANOVA; n = 8 mice per group; ****p < 0.0001. (J) Area under the curve (AUC) analysis of the GTT shown in (I). Data are presented as mean ± SEM; one-way ANOVA. (K) Percent change in body weight between pre-tamoxifen injection and the final day of the experiment. D707A/F, n = 8; D707A/–, n = 13; –/–, n =10. Data are presented as mean ± SEM; one-way ANOVA. (L) Representative H&E staining of pancreatic tissue.

Article Snippet: Insr floxed mice (IR F/F ; The Jackson Laboratory, #006955) were crossed with tamoxifen-inducible CAG-CreERT mice( ) (The Jackson Laboratory, #004682) to generate IR F/F ;CreERT mice, which were subsequently backcrossed for nine generations onto the C57BL/6J background.

Techniques: Binding Assay, Residue, Staining, Injection

Journal: bioRxiv

Article Title: Reprogramming insulin receptor activation with a de novo agonist to overcome severe insulin resistance

doi: 10.64898/2026.05.04.722722

Figure Lengend Snippet: (A) Experimental scheme. Female mice (2–3 months old) were injected with tamoxifen for five consecutive days to delete the floxed WT IR allele and generate D707A/– mice. Mice were fasted for 4 h and injected with PBS, 6 nmol/mouse Humulin or RF-409 via the inferior vena cava (IVC). Liver, skeletal muscle, and adipose tissue were collected at 3, 5, and 7 min after injection, respectively. (B) Representative immunoblot of IR autophosphorylation, AKT phosphorylation (pAKT), and ERK phosphorylation (pERK) in skeletal muscle from IR D707A/– mice. (C) Quantification of immunoblot data shown in (B). Data are presented as mean ± SEM; PBS, n = 4; Humulin, n = 5; RF-409, n = 5; one-way ANOVA. (D) Representative immunoblot of IR autophosphorylation, pAKT, and pERK in adipose tissue from IR D707A/– mice. (E) Quantification of immunoblot data shown in (D). Data are presented as mean ± SEM; PBS, n = 4; Humulin, n = 5; RF-409, n = 5; one-way ANOVA.

Article Snippet: Insr floxed mice (IR F/F ; The Jackson Laboratory, #006955) were crossed with tamoxifen-inducible CAG-CreERT mice( ) (The Jackson Laboratory, #004682) to generate IR F/F ;CreERT mice, which were subsequently backcrossed for nine generations onto the C57BL/6J background.

Techniques: Injection, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: Reprogramming insulin receptor activation with a de novo agonist to overcome severe insulin resistance

doi: 10.64898/2026.05.04.722722

Figure Lengend Snippet: (A) Representative immunoblot of IR protein levels in liver and skeletal muscle from IR D707A/– and IR –/– mice following a 4 h fast. (B) Quantification of immunoblot data shown in (A). Data are presented as mean ± SEM; IR D707A/-, n = 5; IR –/–, n = 7; one-way ANOVA. (C) Representative immunoblot of insulin receptor (IR) autophosphorylation, AKT phosphorylation (pAKT), and ERK phosphorylation (pERK) in liver from IR D707A/– mice. Mice were fasted for 4 h and injected with PBS, Humulin (6 nmol/mouse), or RF-409 via the inferior vena cava (IVC). Liver tissue was collected 3 min after injection. (D) Quantification of immunoblot data shown in (C). Data are presented as mean ± SEM; PBS, n = 4; Humulin, n = 5; RF-409, n = 5; one-way ANOVA. (E) Representative immunoblot of IR autophosphorylation in primary hepatocytes isolated from IR D707A/F;Cre-ERT mice. Cells were treated with 4-hydroxytamoxifen (4-OHT) to delete the floxed WT IR allele and generate IR D707A/– hepatocytes, followed by stimulation with insulin or RF-409 at the indicated concentrations for 10 min. (F) Quantification of immunoblot data shown in (E). Phosphorylation levels were normalized to total protein and expressed relative to the response to 100 nM insulin. Data are presented as mean ± SEM; n = 3 independent experiments; ****p < 0.0001.

Article Snippet: Insr floxed mice (IR F/F ; The Jackson Laboratory, #006955) were crossed with tamoxifen-inducible CAG-CreERT mice( ) (The Jackson Laboratory, #004682) to generate IR F/F ;CreERT mice, which were subsequently backcrossed for nine generations onto the C57BL/6J background.

Techniques: Western Blot, Phospho-proteomics, Injection, Isolation

Journal: bioRxiv

Article Title: Reprogramming insulin receptor activation with a de novo agonist to overcome severe insulin resistance

doi: 10.64898/2026.05.04.722722

Figure Lengend Snippet: (A) Experimental scheme for chronic RF-409 treatment. Male mice (3 months old) were injected with tamoxifen for five consecutive days to delete the floxed WT IR allele and generate IR D707A/– mice. Following the final tamoxifen injection, osmotic pumps releasing PBS or RF-409 were implanted. Body composition was assessed by MRI prior to tamoxifen injection and 14 days after the final tamoxifen injection. (B) Ad libitum blood glucose levels during the treatment period. Data are presented as mean ± SEM; n = 5 mice per treatment; two-way ANOVA; **p < 0.01. (C) Average ad libitum blood glucose levels calculated from (B). Data are presented as mean ± SEM; n = 5 mice per treatment; Welch’s t test. (D) Fasting insulin levels at the end of the treatment period. Data are presented as mean ± SEM; n = 5 mice per treatment; Welch’s t test. (E) Percent change in body weight over the treatment period. Data are presented as mean ± SEM; n = 5 mice per treatment; Welch’s t test. (F) Percent change in fat mass. Data are presented as mean ± SEM; n = 5 mice per treatment; Welch’s t test. (G) Percent change in lean mass. Data are presented as mean ± SEM; n = 5 mice per treatment; Welch’s t test. (H) Epididymal white adipose tissue (eWAT) weight normalized to body weight. Data are presented as mean ± SEM; n = 5 mice per treatment; Welch’s t test. (I) Representative H&E staining (top) and insulin immunostaining (bottom) of pancreatic tissue. (J) Pancreas weight normalized to body weight. Data are presented as mean ± SEM; n = 5 mice per treatment; Welch’s t test. (K) Acinar area quantification. Data are presented as mean ± SEM; n = 5 mice per treatment; Welch’s t test. (L) Representative H&E staining of liver tissue.

Article Snippet: Insr floxed mice (IR F/F ; The Jackson Laboratory, #006955) were crossed with tamoxifen-inducible CAG-CreERT mice( ) (The Jackson Laboratory, #004682) to generate IR F/F ;CreERT mice, which were subsequently backcrossed for nine generations onto the C57BL/6J background.

Techniques: Injection, Staining, Immunostaining