Table 1.
Metabolic Parameters at the end of the study.
Figure 1.
Fisetin reduces the consequences of diabetes in Akita mice.
Male Akita mice were fed control diet or fisetin (0.05%) for 18 weeks beginning at 6 weeks of age. (A) Kidney hypertrophy was assessed by weighing the kidneys and normalizing the kidney weight to the relative weight of the mice. (B) Kidney function was assessed by measuring the albumin/creatinine ratio in the urine. Fisetin reduced diabetes-induced renal hypertrophy and albuminuria. Capillary lumen area, glomerular volume and PAS positive mesangial matrix area were increased in Akita mouse kidneys (E, F: +fisetin) compared with wild type mice (C, D: +fisetin). The increase in capillary lumen area (G) (p<0.05) and glomerular volume in the Akita mouse kidneys was significantly different from controls (H) (p<0.001) and was largely prevented by fisetin. The PAS positive area was also significantly increased in the Akita mouse kidneys (I) (p<0.05) while this difference was not significant in the Akita + fisetin kidneys. All data are mean ± SD of n = 5–7/group. *(p<0.05), **(p<0.01), ***(p<0.001) indicate significantly different from wild type or Akita alone as determined by ANOVA followed by Tukey's post test.
Figure 2.
Fisetin reduces an experimental index of anxiety in Akita mice.
Male Akita mice were fed control diet or fisetin (0.05%) diet for 18 weeks beginning at 6 weeks of age. Locomotor activity was measured in the open field test. Decreased locomotor activity is an indicator of elevated anxiety that is characteristic of diabetics in poor glycemic control. Fisetin restored (A) distance traveled and (B) time ambulatory to nearly control levels. All data are mean ± SD of n = 5–7/group. *(p<0.05) and **(p<0.01) indicate significantly different from wild type or Akita alone as determined by ANOVA followed by Tukey's post test.
Figure 3.
Fisetin reduces biochemical changes in Akita mouse kidneys.
(A) The diabetes-dependent increase in TBARS is prevented by fisetin. Kidneys from wild type, wild type + fisetin, Akita and Akita + fisetin mice were homogenized in PBS containing phosphatase and protease inhibitors and equal amounts of extract were assayed for TBARS. Kidney samples from 6–7 mice were analyzed and the amount of TBARS normalized to protein. (B) The diabetes-dependent up-regulation of osteopontin (OPN) is prevented by fisetin. Kidneys from wild type, wild type+fisetin, Akita and Akita + fisetin mice were homogenized in PBS containing phosphatase and protease inhibitors and equal amounts of protein were analyzed by SDS-PAGE and Western blotting with an antibody to OPN and actin as a loading control. The expression of GCL (C) and the RAGE receptor (D) in kidneys were analyzed by Western blotting as described in (B). Kidney samples from 6–7 mice per group were analyzed and the average intensity of the GCL, RAGE, and OPN bands normalized to actin is shown in the graphs. A representative blot is shown for each. ***(p<0.001), **(p<0.01) and *(p<0.05) indicate significantly different from wild type or Akita alone as determined by ANOVA followed by Tukey's post test.
Figure 4.
Fisetin reduces markers of inflammation in Akita mice.
(A) Male Akita mice were fed control diet or fisetin (0.05%) diet for 18 weeks beginning at 6 weeks of age. C-reactive protein (CRP) and serum amyloid P (SAP) were measured in plasma by Western blotting. Fisetin reduced plasma CRP and SAP levels in the Akita mice to nearly the levels in the control mice. All data are mean ± SD of n = 5–7/group. ***(p<0.001) indicates significantly different from wild type or Akita alone as determined by ANOVA followed by Tukey's post test. (B) Age dependent increases in plasma CRP and urine albumin/creatinine ratio (ACR). Male Akita mice were first tested at 6 weeks of age and then fed control diet or fisetin (0.05%) for 3 weeks prior to re-testing. C-reactive protein (CRP) was measured in plasma by Western blotting. The albumin/creatinine ratio (ACR) in the urine was determined using ELISAs. All data are mean ± SD of n = 8–10/group. **(p<0.01 - 6 week) indicates significantly different from wild type as determined by t-test. **(p<0.01) and ***(p<0.001) indicate significantly different from wild type or Akita alone as determined by ANOVA followed by Tukey's post test.
Figure 5.
Fisetin reduces MG-dependent protein glycation in the kidney and blood.
(A) Kidneys were homogenized in PBS containing phosphatase and protease inhibitors and equal amounts of protein were analyzed by SDS-PAGE and Western blotting with an antibody to MG and actin as a loading control. The entire gel as shown was scanned and the total density normalized to actin. Four to 6 animals were used per group for quantifying band densities with 2 representative kidneys per group shown. (B) Either whole blood was assayed by Western blotting with an anti-MG antibody or kidney tissue with an anti-glyoxylase 1 antibody. The major MG-glycated protein in blood was hemoglobin (shown) and its loading control is total hemoglobin (α and β). Glyoxylase 1 expression was normalized to actin. (C) The activity of glyoxylase 1 was measured and is expressed in units. One unit is the amount of activity required to produce 1 mmole of S-D-lactoylglutathione/min/mg protein. ***(p<0.001), **(p<0.01) and *(p<0.05) indicate significantly different from wild type or Akita alone as determined by ANOVA followed by Tukey's post test. (D) Exponentially dividing HT22 cells were exposed to 5 mM glutamic acid (GA) with or without 2 µM fisetin (Fis) for 8 h. The cells were lysed in SDS sample buffer and equal amounts of protein were analyzed by SDS-PAGE and Western blotting with an antibody to MG and actin as a loading control (n = 3). The kidney mesangial cell line MES13 was grown in 5 mM glucose (glu) (C is control) and some cells transferred to 25 mM glucose plus or minus 10 µM fisetin (Fis) for 8 h. The cells were lysed is SDS sample buffer and equal amounts of protein were analyzed by SDS-PAGE and Western blotting with an antibody to MG and actin as a loading control (n = 3). For both cell lines, the entire gels as shown were scanned and the total densities normalized to actin. Significant differences were determined by ANOVA followed by Tukey's post test. **p<0.01; *** p<0.001.
Figure 6.
Fisetin reduces MG-dependent protein glycation in the cortex.
Brain cortical extracts were prepared and analyzed exactly as for the kidney shown in Fig. 5 with 6 to 8 animals per group. (A) Glyoxylase 1 enzymatic activity. (B) Protein analyzed by SDS-PAGE and Western blotting with an antibody to MG with actin as a loading control. The entire gel as shown was scanned and the total density normalized to actin. (C) Western blotting with anti-GCL and Glo-1 antibodies. In B and C, the data are presented graphically as relative band density of indicated protein over actin. ***(p<0.001), **(p<0.01) and *(p<0.05) indicate significantly different from wild type or Akita alone as determined by ANOVA followed by Tukey's post test.