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A nonrecirculatory liver slice perifusion system has been developed and utilized for investigating glucagon-stimulated glycogenolysis in normal and diabetic states. It has been shown here that slices maintained in this system experience a controlled environment with respect to temperature and pH and remain viable throughout a three-hour experimental period based upon their maintenance of intracellular potassium levels. Although glycogen content falls by 40%, slices exhibit significant glycogenolysis in a dose-response manner upon challenge with glucagon, with maximal concentrations eliciting a 2.2-fold stimulation. This system, which permits nonrecirculatory challenge of liver tissue and subsequent analysis of both intracellular events and overall physiological responses, is extremely useful for examining hormonal mechanisms operating for glucagon, particularly at low concentrations. Using this methodology, liver slices challenged with glucagon exhibit a biphasic dose-response for glycogenolysis. While the second phase parallels cAMP (cyclic adenosine 3':5'-monophosphate) accumulation and cAMP-PK (cAMP-dependent protein kinase) activation, the first is mediated independent of cAMP. Trinitrophenylhistidine-1, homoarginine-12-glucagon (THG), which can antagonize glucagon-stimulated adenylate cyclase, exhibits 50% partial agonist activity for cAMP production and cAMP-PK but full agonism for glycogenolysis. Separation between these events is only two-fold indicating a cAMP-mediated process. [Des-amino-fYRKKE]-glucagon, ([Des-amino-His¹,D-Phe⁴,Tyr⁵,Arg¹²,Lys¹⁷·¹⁸,Glu²¹]-glucagon), another adenylate cyclase antagonist, does not stimulate cAMP or cAMP-PK up to 25 μM yet still elicits glycogenolysis. These results demonstrate that glucagon does indeed stimulate both cAMP-independent as well as cAMP-dependent glycogenolysis in normal liver. In diabetic systems, glucagon elicits attenuated adenylate cyclase activity in liver plasma membranes with reduction in basal activity and extent of stimulation. Maximal stimulation of cAMP production is also reduced by half in liver slices, but in both systems (normal vs. diabetic) EC₅₀ values for cAMP production are identical. Neither THG nor [des-amino-fYRKKE]-glucagon stimulate cAMP production or cAMP-PK in diabetic liver slices. While THG lowers blood glucose levels in vivo, [des-amino-fYRKKE]-glucagon acts as an agonist. These results suggest that the mechanisms which operate for glucagon-stimulated glycogenolysis in normal liver are attenuated in the diabetic state. Furthermore, antagonism of cAMP production alone is insufficient to antagonize glucagon's overall physiological action.

Indiana University-Purdue University Indianapolis (IUPUI) / Sterol Regulatory Element Binding Protein-1 (SREBP-1) is a conserved transcription factor of the basic helix-loop-helix leucine zipper family (bHLH-Zip) that primarily regulates glycolytic and lipogenic enzymes such as L-pyruvate kinase, acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, and mitochondrial glycerol-3-phosphate acyltransferase 1. SREBP-1c activity is higher in the liver of human obese patients, as well as ob/ob and db/db mouse models of obesity and type 2 diabetes, underscoring the role of this transcription factor as a contributor to hepatic steatosis and insulin resistance. Nonetheless, SREBP-1 deficient ob/ob mice, do not display improved glycemia despite a significant decrease in hepatic lipid accumulation, suggesting that SREBP-1 might play a role at regulating carbohydrate metabolism. By silencing SREBP-1 in the liver of normal and type 2 diabetes db/db mice, we showed that indeed, SREBP-1 is needed for appropriate regulation of glycogen synthesis and gluconeogenesis enzyme gene expression. Depleting SREBP-1 activity more than 90%, resulted in a significant loss of glycogen deposition and increased expression of Pck1 and G6pc. Hence, the benefits of reducing de novo lipogenesis in db/db mice were offset by the negative impact on gluconeogenesis and glycogen synthesis. Some studies had also indicated that SREBP-1 regulates the insulin signaling pathway, through regulation of IRS2 and a subunit of the PI3K complex, p55g. To gain insight on the consequences of silencing SREBP-1 on insulin sensitivity, we analyzed the insulin signaling and mTOR pathways, as both are interconnected through feedback mechanisms. These studies suggest that SREBP-1 regulates S6K1, a downstream effector of mTORC1, and a key molecule to activate the synthesis of protein. Furthermore, these analyses revealed that depletion of SREBP-1 leads to reduced insulin sensitivity. Overall, our data indicates that SREBP-1 regulates pathways important for the fed state, including lipogenesis, glycogen and protein synthesis, while inhibiting gluconeogenesis. Therefore, SREBP-1 coordinates multiple aspects of the anabolic response in response to nutrient abundance. These results are in agreement with emerging studies showing that SREBP-1 regulates a complex network of genes to coordinate metabolic responses needed for cell survival and growth, including fatty acid metabolism; phagocytosis and membrane biosynthesis; insulin signaling; and cell proliferation.