A high-concentrate diet induces inflammatory injury via regulating Ca2+/CaMKKβ-mediated autophagy in mammary gland tissue of dairy cows

by Selleckchem | Jul 26 2023

Introduction: Calmodulin-dependent protein kinase β (CaMKKβ) is closely related to Ca2+ concentration. An increase in Ca2+ concentration in the cytoplasm activates CaMKKβ, and activated CaMKKβ affects the activities of AMPK and mTOR and induces autophagy. A high-concentrate diet leads to Ca2+ disorder in mammary gland tissue.

Objectives: Therefore, this study mainly investigated the induction of mammary gland tissue autophagy by a high-concentrate diet and the specific mechanism of lipopolysaccharide (LPS)-induced autophagy in bovine mammary epithelial cells (BMECs).

Material and methods: Twelve mid-lactation Holstein dairy cows were fed with a 40% concentrate diet (LC) and a 60% concentrate diet (HC) for 3 weeks. At the end of the trial, rumen fluid, lacteal vein blood, and mammary gland tissue were collected. The results showed that the HC diet significantly decreased rumen fluid pH, with a pH lower than 5.6 for more than 3 h, indicating successfully induction of subacute rumen acidosis (SARA). The mechanism of LPS-induced autophagy in BMECs was studied in vitro. First, the cells were divided into a Ctrl group and LPS group to study the effects of LPS on the concentration of Ca2+ and autophagy in BMECs. Then, cells were pretreated with an AMPK inhibitor (compound C) or CaMKKβ inhibitor (STO-609) to investigate whether the CaMKKβ-AMPK signaling pathway is involved in LPS-induced BMEC autophagy.

Results: The HC diet increased the concentration of Ca2+ in mammary gland tissue and pro-inflammatory factors in plasma. The HC diet also significantly increased the expression of CaMKKβ, AMPK, and autophagy-related proteins, resulting in mammary gland tissue injury. In vitro cell experiments showed that LPS increased intracellular Ca2+ concentration and upregulated protein expression of CaMKKβ, AMPK, and autophagy-related proteins. Compound C pretreatment decreased the expression of proteins related to autophagy and inflammation. In addition, STO-609 pretreatment not only reversed LPS-induced BMECs autophagy but also inhibited the protein expression of AMPK, thereby alleviating the inflammatory response in BMECs. These results suggest that inhibition of the Ca2+/CaMKKβ-AMPK signaling pathway reduces LPS-induced autophagy, thereby alleviating inflammatory injury of BMECs.

Conclusion: Therefore, SARA may increase the expression of CaMKKβ by increasing Ca2+ levels and activate autophagy through the AMPK signaling pathway, thereby inducing inflammatory injury in mammary gland tissue of dairy cows.

Comments:

The study aimed to investigate the induction of autophagy in mammary gland tissue due to a high-concentrate diet and the specific mechanism of lipopolysaccharide (LPS)-induced autophagy in bovine mammary epithelial cells (BMECs).

To conduct the study, twelve mid-lactation Holstein dairy cows were divided into two groups and fed either a 40% concentrate diet (low concentrate, LC) or a 60% concentrate diet (high concentrate, HC) for a period of three weeks. At the end of the trial, rumen fluid, lacteal vein blood, and mammary gland tissue were collected for analysis.

The results showed that the HC diet caused a significant decrease in rumen fluid pH, indicating the successful induction of subacute rumen acidosis (SARA). Furthermore, the HC diet led to an increase in the concentration of calcium (Ca2+) in mammary gland tissue and elevated levels of pro-inflammatory factors in the plasma. The expression of CaMKKβ (calmodulin-dependent protein kinase β), AMPK (AMP-activated protein kinase), and autophagy-related proteins was significantly increased in the mammary gland tissue, indicating tissue injury.

In vitro cell experiments were conducted to study the mechanism of LPS-induced autophagy in BMECs. The cells were divided into a control group (Ctrl) and an LPS group to observe the effects of LPS on intracellular Ca2+ concentration and autophagy in BMECs. The cells were also pretreated with an AMPK inhibitor (compound C) or a CaMKKβ inhibitor (STO-609) to investigate the involvement of the CaMKKβ-AMPK signaling pathway in LPS-induced autophagy.

The results showed that LPS increased intracellular Ca2+ concentration and upregulated the protein expression of CaMKKβ, AMPK, and autophagy-related proteins in BMECs. Pretreatment with compound C decreased the expression of autophagy-related proteins and reduced inflammation. Pretreatment with STO-609 not only reversed LPS-induced autophagy in BMECs but also inhibited the protein expression of AMPK, thereby alleviating the inflammatory response.

Based on these findings, the study concludes that subacute rumen acidosis (SARA) caused by a high-concentrate diet may increase the expression of CaMKKβ by elevating Ca2+ levels. This, in turn, activates autophagy through the AMPK signaling pathway, leading to inflammatory injury in the mammary gland tissue of dairy cows. Inhibition of the Ca2+/CaMKKβ-AMPK signaling pathway can reduce LPS-induced autophagy and alleviate the inflammatory injury in BMECs.

In summary, the study highlights the relationship between a high-concentrate diet, CaMKKβ-AMPK signaling pathway, autophagy induction, and inflammatory injury in mammary gland tissue. These findings contribute to a better understanding of the mechanisms involved in mammary gland health in dairy cows and may have implications for the management of subacute rumen acidosis and related conditions.