Effects of Glucose on Insulin Release and on Intermediary Metabolism of Isolated Perifused Pancreatic Islets from Fed and Fasted Rats

Abstract

We examined the relationship between glucose-induced insulin release and the intermediary metabolism of islets from fed and fasted rats. Isolated islets were perifused and insulin release measured in the effluent. At various times after switching islets from 2.4 to 8.6 or 14.5 mM glucose or from 2.4 to 14.5 and back to 2.4 mM glucose, islets were quickly frozen, freeze dried, and subsequently analyzed for tissue content of glucose-6-P, fructose-1,6-P2 plus triose-P, Pi, ATP, ADP, 5'-AMP, NADH, NADPH, total NAD, and total NADP using enzymatic fluorometric procedures. When islets from fed rats were exposed to high glucose, there were concomitant increases of insulin release and islet content of glucose-6-P, fructose-1,6-P2 plus triose-P, NADH, and NADPH. During stimulation Pi and 5'-AMP content fell markedly. The total adenine nucleotide content remained constant. Similar secretory and metabolic changes occurred when 1.5 mM Pi was added to the perifusion fluid. When glucose-stimulated islets were switched back to low glucose for 10 min, all substances but fructose-1,6-P2 plus triose-P, 5'-AMP, NADPH, and possibly ATP returned to the prestimulatory level. Starvation of rats for 3 days blocked the secretory response to 8.6 mM glucose. Fructose-1,6-P2 plus triose-P rose but it did not attain the level existing in islets from fed rats. The ratios (ATP)/(5'-AMP) and (ATP)/(Pi)(adp) increased to the values observed in glucose-stimulated islets of fed rats. The metabolic changes in islets from fed rats exposed to high glucose are consistent with an activation of glycolysis occurring concomitantly with stimulated rates of insulin release. This occurs despite the decrease of important activators of glycolysis--Pi and 5'-AMP. The enhanced glycolysis possibly results from P-fructokinase activation by increased fructose-6-P levels. Activation of glycolysis with 8.6 mM glucose was not as pronounced in islets from starved rats. Despite the different secretory response of islets from fet and fasted rats, the changes of phosphorylation state in the islets, in particular, Pi and 5'-AMP levels, were similar.

The Central Role of Glucokinase in Glucose Homeostasis: A Perspective 50 Years After Demonstrating the Presence of the Enzyme in Islets of Langerhans

“…Through the efforts of many laboratories, it was established that calcium and cyclic AMP served as second messengers in stimulus secretion coupling of β-cells (Malaisse et al., 1967; Sussman and Vaughan, 1967; Turtle and Kipnis, 1967; Charles et al., 1973; Zawalich et al., 1975; Wollheim and Sharp, 1981; Liang et al., 1996). This extensive knowledge base allowed Matschinsky and coworkers (Matschinsky and Ellerman, 1968; Trus et al., 1980, 1981; Meglasson et al., 1983; Meglasson and Matschinsky, 1986; Matschinsky, 1990; Ghosh et al., 1991; Matschinsky et al., 1993; Sweet and Matschinsky, 1995; Sweet et al., 1996) to conceptualize a metabolic glucose sensing system involving GCK activity, oxidative phosphorylation, K + ATP channels, and voltage gated calcium channels. Advances in human genetic analysis soon established linkage between the GCK gene and certain forms of MODY (Maturity Onset Diabetes in the Young) by Froguel et al.…”

Are Metabolic Oscillations Responsible for Normal Oscillatory Insulin Secretion?

“…In this regard, it should be noted that most of the adenine nucleotide is in the form of ATP; hence, conversion of a relatively small amount of ADP to ATP can have considerable effect on the ATP/ADP ratio and on the phosphorylation potential (ATP/ADP • P ; ). It may be calculated, based on an ADP content of 3-6 pmol/islet (56,57) and glucose usage of 100 pmol • islet" 1 • h" 1 (37), or an average glycolytic phosphorylation rate of ~3 pmol ADP • islet" 1 • min" 1 , that a pulse of glycolytic phosphorylation equivalent to several minutes' steady flux concentrated into a fraction of a minute could easily consume most of the ADP. It should be noted that it is the cytoplasmic ADP and ATP that are important here for controlling K^p channels as well as downstream effects on the exocytotic process.…”

Pancreatic islet glucose metabolism and regulation of insulin secretion

“…The activity of glucokinase was corrected for partial saturation by ATP based on the K, for ATP measured for islet g l u~o k i n a s e~~ and an estimated cytosolic ATP concentration of 3 mM. 45 Factors other than substrates that may regulate g l u c~k i n a s e~~~~~ were considered to have a negligible effect. Glucokinase activity computed based on glucose saturation plots for glucokinase purified from islets ( 0 ) or insulinomas (m).…”

“…Complete glycolysis results in net formation of ATP and, in islets stimulated by increased glucose, the ATP content is slightly increased; ADP is slightly diminished while AMP and Pi are lowered. 45 It should be noted that these analyses represent total tissue contents and may not reflect the energy state in individual compartments. Also, energy expenditure with secretory events may lower the energy state yet be rapidly compensated so that ATP levels appear unaltered.…”