The Direct Link between Cytosolic pH and Environmental Signals, Insulin Processing, and Secretion in Pancreatic β Cells

Background

Pancreatic β cells respond to glucose fluctuations by regulating insulin processing and secretion. However, how this process is finely tuned within a complex and variable microenvironment, and whether its dysfunction is linked to metabolic diseases, is not fully understood. Here, we demonstrate that the cytosolic pH (pHc) in β cells increases in response to glucose challenges, a change sensed by Smad5 through its nuclear-cytoplasmic shuttling. A deficiency in Smad5 leads to inadequate insulin processing and secretion, resulting in hyperglycemia and impaired glucose tolerance. The role of Smad5 in insulin processing and secretion is attributed to its non-canonical function of regulating secretory granule acidification by modulating V-ATPase activity.

Research Source

This cross-sectional study was led by Yujiang Fang, with team members including Hexi Feng, Bowen Zhang, and Shuwei Zhang, among others, from the Translational Medicine Center at Tongji University in Shanghai, the Institute of Brain and Brain-Like Intelligence, the Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration under the Ministry of Education, and the Shanghai Children’s Medical Center affiliated with Shanghai Jiao Tong University. The research findings were published in Cell Metabolism on June 4, 2024.

Research Process and Methods

Detailed Research Process

Workflow Analysis:

1. Measurement of Cytosolic pH:

   • Researchers measured the cytosolic pH of isolated islet cells through glucose stimulation and BCECF loading. The results showed a significant increase in cytosolic pH in response to high glucose.

2. Smad5 Transmembrane Shuttling and Phosphorylation State:

   • Using GFP-Smad5 reporter mice’s islet cells, the researchers performed time-series analysis and found that GFP-Smad5 shuttled from the nucleus to the cytoplasm within 10-15 minutes under high glucose stimulation. This shuttling phenomenon was confirmed to depend on cytosolic alkalinization and the activity of the nuclear export receptor Crm1 after treatment with exocytosis inhibitors.

3. Glucose Intolerance Induced by High-Fat Diet:

   • HFD-treated mice showed a significantly weakened Smad5 nuclear-cytoplasmic shuttling under high glucose conditions, indicating that the harmful microenvironment induced by HFD reduced β cells’ cytosolic pH. Thus, the researchers hypothesized that cytosolic Smad5 alkalinization could correct HFD-induced glucose intolerance.

4. Genetic Intervention and Alkaline Water Application:

   • Combining Smad5 mutant mice with alkaline water treatment almost completely restored HFD mice’s glucose intolerance symptoms. Alkaline water enhanced glucose-induced cytoplasmic accumulation of Smad5, suggesting it improved the impaired microenvironment and local acidity.

Main Experimental Results

1. Increased Cytosolic pH Induced by High Glucose:

   • The results showed that under high glucose conditions, the cytosolic pH of islet cells significantly increased. Moreover, the cytoplasmic accumulation of Smad5 induced by high glucose depended on the nuclear export receptor Crm1.

2. Insulin Secretion Defects Caused by Smad5 Deficiency:

   • Smad5 knockout mice exhibited elevated fasting blood glucose and impaired glucose tolerance. Fasting blood glucose measurement and glucose tolerance tests further confirmed that this glucose intolerance persisted.

3. V-ATPase Acidification Role:

   • The experiments revealed that the role of V-ATPase in secretory granule acidification is crucial for insulin processing and secretion. Smad5 promotes proton pump function by regulating its activity, ensuring efficient insulin processing.

Conclusions and Significance

1. Scientific Value: The study indicates that cytosolic pH serves as a direct signal linking environmental cues with insulin processing and secretion. Smad5 acts as a “sensor” of pH changes in this process and ensures the proper acidification of secretory granules by regulating V-ATPase activity, thereby ensuring correct insulin processing and secretion.

2. Application Value: The study proposes a new potential therapeutic strategy, which involves correcting insulin processing and secretion defects caused by a high-fat diet through regulating Smad5 nuclear export or cytoplasmic alkalinization, thus improving diabetes symptoms induced by a high-fat diet.

3. Research Highlights:

   • The study is the first to confirm the signaling role of cytosolic pH in human pancreatic β cells.
   • By manipulating Smad5 (e.g., genetic knockout, mutation, or alkaline water treatment), the study demonstrates its critical non-canonical function in regulating insulin processing and secretion.
   • The study proposes a new diabetes treatment method by improving the insulin secretion process through adjustments in cytosolic pH and Smad5 cytoplasmic distribution.

Research Significance and Future Outlook

This research reveals the decisive role of cytosolic pH in insulin processing and secretion, providing new insights into the regulatory mechanisms of β cell function. It shows that Smad5 not only plays a role in the traditional BMP signaling pathway but also has important non-canonical functions. Further exploration of the relationship between Smad5 and other signaling pathways and how these pathways interact under pathological conditions (such as obesity and diabetes) is necessary. Through in-depth research, this may provide a reliable basis for developing new diabetes treatment strategies.