Fig 1.
Urtica dioica extract partially rescues FFA induced reduction in adiponectin expression.
Fully differentiated 3T3-L1 adipocytes were incubated with indicated doses of palmitate for 72 hours and media (A) and whole cell extracts (B) were examined for adiponectin expression. Adipocytes were also treated with palmitate for 72 hours in the presence or absence of UT (Urtica dioica) for 72 hours and media (C) and whole cell extracts (D) were examined for adiponectin expression. Samples were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the respective antibodies before detection by enhanced chemiluminescence. Each panel represents an experiment independently repeated three times on independent batches of adipocytes. ADPN expression in media and cells was normalized by the corresponding β-Actin expression in cells. Treatments were compared by ANOVA followed by the Tukeys test of multiple comparisons. *denotes significant difference between the control treatments and FFA treatment (P<0.05). #denotes significant difference between the FFA and FFA + UT treatments (P<0.05)
Fig 2.
FFAs induce ceramidase protein expression in a dose dependent manner but do not increase ceramidase activity in adipocytes.
Fully differentiated 3T3-L1 adipocytes were incubated with indicated doses of palmitate for 72 hours and (A) cell extracts were examined for acid ceramidase and neutral ceramidase expression. Samples were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with the respective antibodies before detection by enhanced chemiluminescence. (B) Neutral ceramidase activity was determined by fluorometric quantification of the fluorescent sphingosine generated in the cells after 24 hours incubation with C18 NBD ceramide. Acid ceramidase was similarly determined after incubation with C12 NBD ceramide. Treatments were compared by ANOVA followed by Tukeys test of multiple comparisons. Each panel represents an experiment independently repeated three times on independent groups of adipocytes. *denotes significant difference between the control treatments and FFA treatment (P<0.05).
Fig 3.
Urtica dioica extract does not affect ceramidase expression but enhances ceramidase activity and reduces ceramide levels in adipocytes.
Fully differentiated 3T3-L1 adipocytes were incubated with palmitate (800 uM) and UT extract (5ug/ml) for 72 hours and (A) Cell lysates were used for western blot analysis. (B) Acid ceramidase activity was determined by fluorometric quantification of the fluorescent sphingosine generated in the cells after 24 hours incubation with C12 NBD ceramide. Neutral ceramidase was similarly determined after incubation with C18 NBD ceramide. (C) Total ceramides were quantified by tandem mass spectrometry (LCMS/MS). Each panel represents an experiment independently repeated three times on different batches of adipocytes. Treatments were compared by ANOVA followed by Tukeys test of multiple comparisons. *denotes significant difference between the FFA and FFA + UT treatments (P<0.05). #denotes significant difference between the control treatments and UT only treatment (P<0.05).
Fig 4.
Adiponectin expression is required for Urtica dioica L. to enhance ceramidase activity and decrease ceramide accumulation.
(A) Fully differentiated 3T3-L1 adipocytes were transfected with either a non-targeting siRNA or adiponectin siRNA and treated 48 hours later with palmitate (800uM) and/or UT extract (5ug/ml) for 72 hours. Cell lysates were used for western blot analysis to examine adiponectin expression. (B) Total ceramides were quantified by tandem mass spectrometry (LCMS/MS). (C) Acid ceramidase activity was determined by fluorometric quantification of the fluorescent sphingosine generated in the cells after 24 hours incubation with C12 NBD ceramide. (D) Neutral ceramidase was similarly determined after incubation with C18 NBD ceramide. Each panel represents an experiment independently repeated three times on different batches of adipocytes. Treatments were compared by ANOVA followed by Tukeys test of multiple comparisons.*denotes significant difference between the FFA and FFA + UT treatments (P<0.05) in adipocytes transfected with non-targeting siRNA (P<0.05). #denotes significant difference between the control treatments and UT only treatment in adipocytes transfected with non-targeting siRNA (P<0.05).
Fig 5.
Urtica dioica extract enhances Akt phosphorylation in FFA treated cells in absence of Adiponectin.
Fully differentiated 3T3-L1 adipocytes were incubated with indicated doses of (A) palmitate or (B) palmitate (800uM) and UT. Prior to harvest, adipocytes were stimulated with vehicle or 100 nM insulin for 8 minutes before collection in lysis buffer. Cell lysates were used for western blot analysis to examine Akt serine phosphorylation. Each panel represents an experiment independently repeated three times on different batches of adipocytes. Treatments were compared by ANOVA followed by the Tukeys test of multiple comparisons. #denotes significant difference between the control and FFA only or UT only treatments (P<0.05). *denotes significant difference between the FFA and FFA + UT treatments (P<0.05). (C) Fully differentiated 3T3-L1 adipocytes were transfected with either a non-targeting siRNA or adiponectin siRNA and treated 48 hours later with palmitate (800uM) and/or UT extract (5ug/ml) for 72 hours. Prior to harvest, adipocytes were stimulated with 100 nM insulin for 8 minutes before collection in lysis buffer. Cell lysates were used for western blot analysis to examine Akt serine phosphorylation. Each panel represents an experiment independently repeated three times on different batches of adipocytes. Treatments were compared by ANOVA followed by the Tukeys test of multiple comparisons. *denotes significant difference between the FFA and FFA + UT treatments in adipocytes transfected with non-targeting siRNA. #denotes significant difference between the FFA and FFA + UT treatments in adipocytes with adiponectin knockdown (P<0.05).