Glucagon changes substrate preference in gluconeogenesis.

Area of Science:

• Biochemistry
• Metabolic Regulation
• Diabetes Pathophysiology

Background:

• Fasting hyperglycemia in diabetes mellitus stems from unregulated glucagon secretion, driving gluconeogenesis (GNG).
• Previous studies on GNG substrates in hepatocytes used limited substrates and non-physiologic concentrations.
• Understanding substrate utilization is key to managing hyperglycemia.

Purpose of the Study:

• To investigate the relative contributions of different substrates (glycerol, pyruvate/lactate, glutamine) to glucose production in primary hepatocytes under fasting conditions.
• To determine how glucagon stimulation affects the utilization of these substrates in gluconeogenesis.
• To elucidate the role of glycerol as a carbon donor in hepatic gluconeogenesis.

Main Methods:

• Cultured primary hepatocytes were treated with mixtures of U-¹³C- or 2-¹³C-labeled substrates (pyruvate/lactate, glutamine, glycerol) at fasting concentrations.
• ¹³C labeling was used to trace substrate incorporation into newly synthesized glucose.
• Metabolic flux analysis and *in vivo* studies in a PKA-activation mouse model were employed.

Main Results:

• In the absence of glucagon, 80% of glucose produced incorporated labeled glycerol, indicating its high contribution to GNG.
• Glucagon stimulation increased glycerol and pyruvate/lactate contributions by 1.6- and 1.8-fold, respectively, but increased glutamine's contribution significantly (6.4-fold).
• Metabolic flux analysis confirmed glycerol as the predominant carbon source for glucose production by hepatocytes. *In vivo* studies showed lactate carbons originated from glycerol, not vice versa.

Conclusions:

• Glycerol is a major, and often primary, carbon donor for hepatic gluconeogenesis, particularly under fasting conditions.
• While glucagon significantly enhances glutamine's role in GNG, glycerol remains a critical substrate.
• The complex substrate utilization in GNG suggests hepatic glucagon action involves multiple mechanisms, not a single pathway.