Milk-derived tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) promote adipocyte differentiation and inhibit inflammation in 3T3-F442A cells

Abstract

Milk derived tripeptides IPP (Ile-Pro-Pro) and VPP (Val-Pro-Pro) have shown promise as anti-hypertensive agents due to their inhibitory effects on angiotensin converting enzyme (ACE). Due to the key inter-related roles of hypertension, chronic inflammation and insulin resistance in the pathogenesis of metabolic syndrome, there is growing interest in investigating established anti-hypertensive agents for their effects on insulin sensitivity and inflammation. In this study, we examined the effects of IPP and VPP on 3T3-F442A murine pre-adipocytes, a widely used model for studying metabolic diseases. We found that both IPP and VPP induced beneficial adipogenic differentiation as manifested by intracellular lipid accumulation, upregulation of peroxisome proliferator-activated receptor gamma (PPARγ) and secretion of the protective lipid hormone adiponectin by these cells. The observed effects were similar to those induced by insulin, suggesting potential benefits in the presence of insulin resistance. IPP and VPP also inhibited cytokine induced pro-inflammatory changes such as reduction in adipokine levels and activation of the nuclear factor kappa B (NF-κB) pathway. Taken together, our findings suggest that IPP and VPP exert insulin-mimetic adipogenic effects and prevent inflammatory changes in adipocytes, which may offer protection against metabolic disease.

Fig 1. IPP and VPP induce lipid accumulation in 3T3-F442A cells.

3T3-F442A cells were incubated in presence of insulin (10 μg/mL), IPP (50 μM) or VPP (50 μM) for 72 hr. (A) For one study, the cells were fixed, stained with the neutral lipid-specific dye LipidTox and visualized under fluorescence microscopy. A set of representative images are shown. (B) For another set of experiments, the cells were lysed and their lipid contents were estimated by a biochemical assay. Data were presented as mean±SEM of 3–4 independent experiments. * and ** indicate p<0.05 and p<0.01 respectively compared to the untreated control (Untr).

Fig 2. IPP and VPP promote expression of the adipocyte differentiation markers PPARγ and adiponectin in 3T3-F442A cells.

3T3-F442A cells were incubated in presence of insulin (10 μg/mL), IPP (50 μM) or VPP (50 μM) for 72 hr. (A) The cells were lysed and western blotting of the lysates was performed with antibodies against PPARγ and α-tubulin (loading control). A set of representative images (including cropped images obtained from the same membrane) was shown. (B) The cell-free culture supernatants were collected and analyzed by ELISA to determine levels of adiponectin. Data were presented as mean±SEM of 5 independent experiments. * and *** indicate p<0.05 and p<0.001 compared to the untreated control (Untr) respectively.

Fig 3. IPP and VPP increase the protein levels of adipocyte differentiation regulators c-Jun and C/EBPα.

3T3-F442A cells were incubated in presence of insulin (10 μg/mL), IPP (50 μM) or VPP (50 μM) for 72 hr. The cells were lysed and western blotting of the lysates was performed with antibodies against c-Jun (A), C/EBPα (B) and α-tubulin (loading control). A set of representative images was shown. Data were presented as mean±SEM of 4–6 independent experiments. * and ** indicate p<0.05 and p<0.01 respectively, compared to the untreated control (Untr). # and ## indicate p<0.05 and p<0.01 respectively, compared to the insulin treated cells.

Fig 4. IPP and VPP inhibit TNFα mediated activation of the pro-inflammatory NF-κB pathway downstream of IκB degradation.

3T3-F442A cells were incubated for 48 hr in presence of insulin (10 μg/mL) to induce differentiation. Cells were then washed and further incubated for 30 min with pro-inflammatory cytokine TNFα (10 ng/mL) with/without addition of IPP (50 μM) or VPP (50 μM). Afterwards, the cells were lysed and western blotting of the lysates was performed to determine (A) IκBα degradation (using antibodies against IκBα and the loading control, α-tubulin) and (B) p65 phosphorylation (using antibodies against phosphorylated and total p65). A set of representative images was shown. Data were presented as mean±SEM of 4 independent experiments. All data were normalized to the values from untreated (i.e. undifferentiated) cells. * indicates p<0.05 compared to the TNFα treated cells. NS means: not significant (compared to TNFα treated group).

Fig 5. IPP and VPP prevent TNFα mediated loss of adiponectin release from insulin-differentiated 3T3-F442A cells.

3T3-F442A cells were incubated for 48 hr in presence of insulin (10 μg/mL) to induce differentiation. Cells were then washed and further incubated for 24 hr with the pro-inflammatory cytokine TNFα (10 ng/mL) with/without addition of IPP (50 μM) or VPP (50 μM). At the end of this incubation period, the cell-free supernatants were collected and analyzed for their adiponectin content by ELISA. Data were presented as mean±SEM of 4 independent experiments. * indicates p<0.05 compared to the insulin alone (Alone). NS means: not significant (compared to Alone).