Fig 1: Summary. When insulin sensitivity is improved at the level of the IR in podocytes, either through IR over-expression or PTP1B knockdown, podocytes are protected from the development of ER stress. However, knockdown of PTEN, which improves insulin sensitivity through the PI3K-Akt pathway downstream of insulin, potentiates ER stress. Inhibition of mitogen-activated protein kinase kinase (MEK) or extracellular signal-regulated kinase (ERK) protects podocytes from ER stress. (IRS, insulin receptor substrate).
Fig 2: The amount of PTP1B in podocytes positively correlates with ER stress. WT and PTP1B kd cells untreated or treated with 300 µM palmitate were stained for CHOP, PTP1B and with DAPI. (a) Immunofluorescent staining of wt and PTP1B kd cells for PTP1B. (b) Frequency distribution plot of unadjusted PTP1B intensity values for single cells sorted into 50-AFU bins. (c) Nuclear PTP1B and CHOP paired single cell values were sorted based on the amount of PTP1B into 50-AFU bins. WT and PTP1B kd cells were combined in silico to yield a wide range of PTP1B intensity values for cells untreated or palmitate-treated. Within each bin, the mean CHOP value was calculated and plotted. Linear regression analysis showed that both slopes were significantly non-zero, p < 0.0001. R2 = 0.92 for untreated podocytes and 0.95 for palmitate-treated. Two-way ANOVA, effect of PTP1B expression ****p < 0.0001, effect of palmitate treatment ****p < 0.0001, with a significant interaction between PTP1B expression and palmitate treatment ****p < 0.0001. Bonferroni post-hoc comparison comparing row means (CHOP expression without or with palmitate treatment for each value of PTP1B) revealed means were significantly different (****p < 0.0001) for each value of PTP1B.
Fig 3: Knockdown of PTP1B protects against ER stress. (a) Representative western blot with densitometric quantification normalised to GAPDH demonstrating PTP1B knockdown of 56% in the PTP1B kd cell line compared with wt cells (n = 3). Unpaired t test **p = 0.005. Quantification of PTP1B in cells treated with scrambled (scr) shRNA also shown. (Full blot shown in Supplementary Fig. 3). (b) Insulin sensitivity monitored by pAkt immunostaining in cell nuclei following 10 min of 10-10-10-6 M insulin stimulation (n = 4). Data expressed as the percentage of cells positive for pAkt in the nucleus. Two-way ANOVA, effect of PTP1B p < 0.0001****, insulin ***p = 0.0002, but no significant interaction between the effect of PTP1B and insulin concentration. A Bonferroni post-hoc comparison revealed statistical significance at 10-7 M insulin (*). (c) ATF6-driven luciferase activity for wt and PTP1B kd podocytes treated with diabetic media relative to normal growth media. Unpaired t test with Welch’s correction (n = 3), *p < 0.05. (d) ERSE-driven luciferase activity for wt and PTP1B kd podocytes treated with diabetic media relative to normal growth media. Unpaired t test with Welch’s correction (n = 3). (e) CHOP response in wt and PTP1B kd podocytes for cells treated with or without diabetic media (‘D’), (n = 3). Comparison of wt and PTP1B kd, unpaired t test with Welch’s correction; paired t test for comparison of wt and wt + D p = 0.0091 (**), wt + D and PTP1B kd p = 0.0006 (***), PTP1B kd and PTP1B kd + D (ns). (f) Caspase 3/7 activation in wt and PTP1B kd podocytes following 24 hr palmitate treatment (n = 3). Two-way ANOVA, effect of PTP1B kd not significant, effect of palmitate ****p < 0.0001.
Fig 4: Long-term ß-glucan supplementation improved PTP1B-IRS-pAKT-pGSK3ß-pTau and synapse in the hippocampus of HFFD-induced obese mice. a The protein level of PTP1B in the hippocampus (n = 6). b–d Protein levels of p-IRS-1/IRS-1, p-Akt/Akt, and p-GSK3ß/GSK3ß (n = 6). e The protein level of p-Tau/Tau in the hippocampus (n = 4–6). f The ultrastructure of synapses on the electron micrograph in the hippocampus CA1 region of mice fed with different diets (scale bar 500 nm). The enlarged images of the second row were from the first row in the area indicated with a dotted line box (scale bar 250 nm). g and h Image analysis of the thickness of PSD and the width of the synaptic cleft (n = 3). PSD, postsynaptic density; SC, synaptic cleft; SV, synaptic vesicle. i and j The protein levels of SYN and PSD95. Values are mean ± SEM. *p < 0.05 vs. control (Con). #p < 0.05 vs. high-fat and fiber-deficient (HFFD)
Fig 5: Diet rich in microbiota-accessible carbohydrate inhibited PTP1B and improved synaptic signaling molecules in the hippocampus of diet-induced obese mice. a Protein level of PTP1B in the hippocampus (n = 6). b–d Protein levels of p-IRS-1/IRS-1, p-Akt/ Akt, and p-GSK3ß/ GSK3ß (n = 6). e Protein level of p-Tau/Tau in the hippocampus (n = 4). f Phosphorylation cascade of insulin IRS-pAKT-pGSK3ß-pTau synapse signaling. Values are mean ± SEM. *p < 0.05 vs. Control (Con). #p < 0.05 vs. high-fat and fiber-deficient (HF-FD)
Supplier Page from Abcam for Anti-PTP1B antibody