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01.01.2012 | Article | Ausgabe 1/2012

Diabetologia 1/2012

Amylin and leptin activate overlapping signalling pathways in an additive manner in mouse GT1-7 hypothalamic, C2C12 muscle and AML12 liver cell lines

Zeitschrift:
Diabetologia > Ausgabe 1/2012
Autoren:
H.-S. Moon, J. P. Chamberland, C. S. Mantzoros
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1007/​s00125-011-2332-0) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Abstract

Aims/hypothesis

It has been suggested that amylin amplifies leptin’s effects and affects energy homeostasis synergistically with leptin in animals and humans. However, no previous study has reported on amylin and leptin signalling in hypothalamic, muscle and liver cells.

Methods

Leptin and amylin signalling studies were performed in vitro in mouse GT1-7 hypothalamic, C2C12 muscle and AML12 liver cell lines.

Results

Treatment of mouse GT1-7 and C2C12 cells with leptin or amylin increased signal transducer and activator of transcription 3 (STAT3) phosphorylation in a dose- and time-dependent manner. In mouse AML12 cells, leptin and amylin produced a biphasic response of STAT3 activity. Importantly, all leptin and amylin signalling pathways were saturable at leptin and amylin concentrations of ∼100 and ∼50 to 100 ng/ml, respectively. Leptin and amylin in combination activated STAT3, AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK) 1/2 and Akt signalling pathways in an additive manner, effects that were abolished under endoplasmic reticulum (ER) stress. Leptin, but not amylin, increased IRS-1 phosphorylation in GT1-7 hypothalamic, but not in C2C12 muscle and AML12 liver cell lines.

Conclusions/interpretation

Our data suggest that leptin and amylin have overlapping and additive, but not synergistic, effects in the activation of intracellular signalling pathways. ER stress may induce leptin and amylin resistance in hypothalamic, muscle and liver cell lines. These novel insights into the mode of action of leptin and amylin suggest that these hormones may play an additive role in regulating energy homeostasis in humans.

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Zusatzmaterial
ESM Fig. 1 Activation of STAT3 signaling by low physiological concentrations of leptin and amylin in mouse GT1-7 hypothalamic, C2C12 muscle, and AML12 liver cell lines - The cell culture was performed as described in detail in “Methods”. (A–B) The cells were incubated with leptin and/or amylin at indicated concentrations for 45 min. All density values for each protein band of interest are expressed as a fold increase. All data were analyzed using one-way ANOVA followed by post-hoc test for multiple comparisons. Values are means (n = 3) ± SD. Means with different letters are significantly different, p < 0.05. C: control (PDF 61 kb)
ESM Fig. 2 Leptin and amylin alone or in combination increase cell proliferation in mouse GT1-7 hypothalamic, C2C12 muscle, and AML12 liver cell lines - The cell culture was performed as described in detail in “Methods”. The cells were incubated with 50 ng/ml of leptin, 20 ng/ml of amylin and/or leptin + amylin for 36 hr and cell proliferation assay was then performed as described in detail in “Methods”. All data were analyzed using one-way ANOVA followed by post-hoc test for multiple comparisons. Values are means (n = 3) ± SD. Means with different letters are significantly different, p < 0.05. C: control, L: leptin, A: amylin, L + A: leptin + amylin (PDF 17 kb)
ESM Fig. 3 Activation of eIF2 signaling by ER stress in mouse GT1-7 hypothalamic,C2C12 muscle, and AML12 liver cell lines–The cell culture was performed as described in detail in “Methods”. The cells were treated with TUN (3 ug/ml) and/or DTT (1 mM) for 5 hr. All density values for each protein band of interest are expressed as a fold increase. All data were analyzed using one-way ANOVA followed by post-hoc test for multiple comparisons. Values are means (n = 3) ± SD. Means with different letters are significantly different, p < 0.05. C: control (PDF 89 kb)
ESM Fig. 4 Activation of IRS-1 and IRS-2 signaling by administration of leptin and amylin alone or in combination in mouse GT1-7 hypothalamic, C2C12 muscle, and AML12 liver cell lines - The cell culture was performed as described in detail in “Methods”. (A–B) The cells were incubated with 50 ng/ml of leptin, 20 ng/ml of amylin and/or leptin + amylin for 45 min. Cell lysates were immunoprecipitated as described in detail in “Methods”. All density values for each protein band of interest are expressed as a fold increase. All data were analyzed using one-way ANOVA followed by post-hoc test for multiple comparisons. Values are means (n = 3) ± SD. Means with different letters are significantly different, p < 0.05. C: control, L: leptin, A: amylin, L + A: leptin + amylin (PDF 57 kb)
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