Glucose-dependent insulinotropic polypeptide induces cytokine expression, lipolysis, and insulin resistance in human adipocytes.

Obesity-related insulin resistance is linked to a chronic state of systemic and adipose tissue-derived inflammation. Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone also acting on adipocytes. We investigated whether GIP affects inflammation, lipolysis, and insulin resistance in human adipocytes. Human subcutaneous preadipocyte-derived adipocytes, differentiated in vitro, were treated with human GIP to analyze mRNA expression and protein secretion of cytokines, glycerol, and free fatty acid release and insulin-induced glucose uptake. GIP induced mRNA expression of IL-6, IL-1ß, and the IL-1 receptor antagonist IL-1Ra, whereas TNFα, IL-8, and monocyte chemotactic protein (MCP)-1 remained unchanged. Cytokine induction involved PKA and the NF-κB pathway as well as an autocrine IL-1 effect. Furthermore, GIP potentiated IL-6 and IL-1Ra secretion in the presence of LPS, IL-1ß, and TNFα. GIP induced lipolysis via activation of hormone-sensitive lipase and was linked to NF-κB activation. Finally, chronic GIP treatment impaired insulin-induced glucose uptake possibly due to the observed impaired translocation of glucose transporter GLUT4. In conclusion, GIP induces an inflammatory and prolipolytic response via the PKA -NF-κB-IL-1 pathway and impairs insulin sensitivity of glucose uptake in human adipocytes.

Both inflammatory and classical lipolytic pathways are involved in lipopolysaccharide-induced lipolysis in human adipocytes.

High fat diet-induced endotoxaemia triggers low-grade inflammation and lipid release from adipose tissue. This study aims to unravel the cellular mechanisms leading to the lipopolysaccharide (LPS) effects in human adipocytes. Subcutaneous pre-adipocytes surgically isolated from patients were differentiated into mature adipocytes in vitro. Lipolysis was assessed by measurement of glycerol release and mRNA expression of pro-inflammatory cytokines were evaluated by real-time PCR. Treatment with LPS for 24 h induced a dose-dependent increase in interleukin (IL)-6 and IL-8 mRNA expression. At 1 µg/ml LPS, IL-6 and IL-8 were induced to 19.5 ± 1.8-fold and 662.7 ± 91.5-fold (P < 0.01 vs basal), respectively. From 100 ng/ml to 1 µg/ml, LPS-induced lipolysis increased to a plateau of 3.1-fold above basal level (P < 0.001 vs basal). Co-treatment with inhibitors of inhibitory kappa B kinase kinase beta (IKKß) or NF-κB inhibited LPS-induced glycerol release. Co-treatment with the protein kinase A (PKA) inhibitor H-89, the lipase inhibitor orlistat or the hormone-sensitive lipase (HSL) inhibitor CAY10499 abolished the lipolytic effects of LPS. Co-treatment with the MAPK inhibitor, U0126 also reduced LPS-induced glycerol release. Inhibition of lipolysis by orlistat or CAY10499 reduced LPS-induced IL-6 and IL-8 mRNA expression. Induction of lipolysis by the synthetic catecholamine isoproterenol or the phosphodiesterase type III inhibitor milrinone did not alter basal IL-6 and IL-8 mRNA expression after 24 treatments whereas these compounds enhanced LPS-induced IL-6 and IL-8 mRNA expression. Both the inflammatory IKKß/NF-κB pathway and the lipolytic PKA/HSL pathways mediate LPS-induced lipolysis. In turn, LPS-induced lipolysis reinforces the expression of pro-inflammatory cytokines and, thereby, triggers its own lipolytic activity.

Targeting AMP-activated protein kinase in adipocytes to modulate obesity-related adipokine production associated with insulin resistance and breast cancer cell proliferation.

BACKGROUND: Adipokines, e.g. TNFα, IL-6 and leptin increase insulin resistance, and consequent hyperinsulinaemia influences breast cancer progression. Beside its mitogenic effects, insulin may influence adipokine production from adipocyte stromal cells and paracrine enhancement of breast cancer cell growth. In contrast, adiponectin, another adipokine is protective against breast cancer cell proliferation and insulin resistance.AMP-activated protein kinase (AMPK) activity has been found decreased in visceral adipose tissue of insulin-resistant patients. Lipopolysaccharides (LPS) link systemic inflammation to high fat diet-induced insulin resistance. Modulation of LPS-induced adipokine production by metformin and AMPK activation might represent an alternative way to treat both, insulin resistance and breast cancer. METHODS: Human preadipocytes obtained from surgical biopsies were expanded and differentiated in vitro into adipocytes, and incubated with siRNA targeting AMPKalpha1 (72 h), LPS (24 h, 100 µg/ml) and/or metformin (24 h, 1 mM) followed by mRNA extraction and analyses. Additionally, the supernatant of preadipocytes or derived-adipocytes in culture for 24 h was used as conditioned media to evaluate MCF-7 breast cancer cell proliferation. RESULTS: Conditioned media from preadipocyte-derived adipocytes, but not from undifferentiated preadipocytes, increased MCF-7 cell proliferation (p < 0.01). Induction of IL-6 mRNA by LPS was reduced by metformin (p < 0.01), while the LPS-induced mRNA expression of the naturally occurring anti-inflammatory cytokine interleukin 1 receptor antagonist was increased (p < 0.01). Silencing of AMPKalpha1 enhanced LPS-induced IL-6 and IL-8 mRNA expression (p < 0.05). CONCLUSIONS: Adipocyte-secreted factors enhance breast cancer cell proliferation, while AMPK and metformin improve the LPS-induced adipokine imbalance. Possibly, AMPK activation may provide a new way not only to improve the obesity-related adipokine profile and insulin resistance, but also to prevent obesity-related breast cancer development and progression.

Metformin counters both lipolytic/inflammatory agents-decreased hormone sensitive lipase phosphorylation at Ser-554 and -induced lipolysis in human adipocytes.

OBJECTIVE: To study the effects of metformin on lipolysis and hormone sensitive lipase (HSL) phosphorylation in human adipocytes treated with lipolytic and inflammatory agents. METHODS: Lipolysis and phosphorylation status of HSL were assessed in subcutaneous pre-adipocytes surgically isolated from patients and differentiated into mature adipocytes in vitro. RESULTS: Stimulation for 1 h with forskolin, isoproterenol and IBMX or stimulation for 24 h with LPS, IL-1ß and TNF-α increased lipolysis (p < 0.05 vs. basal). The phosphorylation of HSL at Ser-554 was decreased while the Ser-552 phosphorylation was increased. Pre-incubation with metformin (24 h, 1 mM) inhibited forskolin-, isoproterenol-, IBMX-, LPS-, IL-1ß- and TNF-α-induced glycerol release and prevented p(Ser554)HSL decrease and p(Ser-552)HSL increase due to lipolytic and inflammatory agents. AMPKα1 is involved in metformin-induced HSL phosphorylation at Ser-552. CONCLUSION: Phosphorylation of HSL at Ser-554 inversely correlates with lipolysis and HSL phosphorylation at Ser552 in human adipocytes.

Glucose-dependent insulinotropic polypeptide (GIP) induces calcitonin gene-related peptide (CGRP)-I and procalcitonin (Pro-CT) production in human adipocytes.

CONTEXT: Increased plasma levels of glucose-dependent insulinotropic polypeptide (GIP), calcitonin CT gene-related peptide (CGRP)-I, and procalcitonin (Pro-CT) are associated with obesity. Adipocytes express functional GIP receptors and the CT peptides Pro-CT and CGRP-I. However, a link between GIP and CT peptides has not been studied yet. OBJECTIVE: The objective of the study was the assessment of the GIP effect on the expression and secretion of CGRP-I and Pro-CT in human adipocytes, CGRP-I and CT gene expression in adipose tissue (AT) from obese vs. lean subjects, and plasma levels of CGRP-I and Pro-CT after a high-fat meal in obese patients. DESIGN AND PARTICIPANTS: Human preadipocyte-derived adipocytes, differentiated in vitro, were treated with GIP. mRNA expression and protein secretion of CGRP-I and Pro-CT were measured. Human CGRP-I and CT mRNA expression in AT and CGRP-I and Pro-CT plasma concentrations were assessed. RESULTS: Treatment with 1 nm GIP induced CGRP-I mRNA expression 6.9 ± 1.0-fold (P < 0.001 vs. control) after 2 h and CT gene expression 14.0 ± 1.7-fold (P < 0.001 vs. control) after 6 h. GIP stimulated CGRP-I secretion 1.7 ± 0.2-fold (P < 0.05 vs. control) after 1 h. In AT samples of obese subjects, CGRP-I mRNA expression was higher in sc AT (P < 0.05 vs. lean subjects), whereas CT expression was higher in visceral AT (P < 0.05 vs. lean subjects). CGRP-I plasma levels increased after a high-fat meal in obese patients. CONCLUSION: GIP induces CGRP-I and CT expression in human adipocytes. Therefore, elevated Pro-CT and CGRP-I levels in obesity might result from GIP-induced Pro-CT and CGRP-I release in AT and might be triggered by a high-fat diet. How these findings relate to the metabolic complications of obesity warrants further investigations.