Introduction
Renal gluconeogenesis is mainly regulated by acidosis and starvation. Its proportion increases from 10% of total gluconeogenesis under feeding conditions to 40–50% after starvation [
1‐
3]. The main substrates for renal gluconeogenesis are lactate (from the muscle) and glutamine (from throughout the body) [
3‐
5]. Glutamine is converted to glutamate and then to α-ketoglutarate in the mitochondria by a deaminase reaction, producing two ammonia molecules in a process called ammoniagenesis. Then, α-ketoglutarate enters the tricarboxylic acid (TCA) cycle as a source of gluconeogenesis [
6]. Phosphoenol pyruvatecarboxykinase (PEPCK) is activated by acidosis in the kidneys of rats fed ammonium chloride, indicating that acidosis enhances renal gluconeogenesis [
6‐
8].
Vacuolar H
+-adenosine triphosphatase (ATPase) is expressed in the brush border membrane of the proximal tubules and in the intercalated cells of the collecting duct to play an important role in the acid–base balance [
9‐
12] as well as in endocytosis by the acidification of endocytic vesicles [
12‐
14]. The proximal convoluted tubules are the most important sites for both renal ammoniagenesis and gluconeogenesis, and acidosis promotes the urinary excretion of ammonium through the activation of H
+-ATPase and gluconeogenesis [
6‐
8]. A specific inhibitor of H
+-ATPase, bafilomycin (BFM) B1, was discovered from Streptomyces by Nobel Laureate Professor Satoshi Omura as setamycin in 1981 [
15]. We hypothesized that inhibition of H
+-ATPase by BFM B1 may inhibit renal gluconeogenesis and could reduce fasting plasma glucose level under starvation condition.
Discussion
In this study, we demonstrated that the H
+-ATPase activity and ammoniagenesis are enhanced in diabetic rats. Thus far, the H
+-ATPase activity in the kidney has not been studied in DM models. Contrary to our results, the H
+-ATPase activity in the microvascular endothelial cells was found to be decreased in a diabetic model [
18]. Given that prorenin receptors stimulate the H
+-ATPase activity in the renal tubular cells [
19] and that the level of prorenin receptors is increased in the diabetic kidney [
20], our finding of enhanced H
+-ATPase activity in the kidneys of diabetic rats seems reasonable.
The hypoglycemic effect of the blockade of H
+-ATPase by BFM B1 is quite striking, as it is effective in an animal model of STZ-induced insulinopenia. Thus, the anti-diabetic effect was not ascribed to insulin secretion or insulin sensitivity, even though the reduced insulin sensitivity evaluated by KITT value in STZ-diabetic rat was increased by BFM B1 treatment. Also plasma glucagon level and hepatic gluconeogenesis did not change significantly by BFM B1 treatment, so the main mechanism of the hypoglycemic effect of BFM may be dependent upon renal gluconeogenesis under starvation condition. More than 30 years ago, it was reported that vanadate reduced plasma glucose levels in diabetic rats through the inhibition of increased hepatic and renal levels of PEPCK, tyrosine aminotransferase, and glucokinase [
21‐
24]. Interestingly, vanadate is an inhibitor of P-type ATPase, but also inhibits H
+-ATPase [
25‐
27] and this could be related to the hypoglycemic effect of vanadate. Furthermore, chloroquine inhibits H
+-ATPase and glucose formation in the liver and the kidney through the suppression of PEPCK and G-6-Pase [
28]. The hypoglycemic effect of chloroquine is blocked by NH
4Cl [
28], which induces metabolic acidosis and stimulates ammoniagenesis and H
+-ATPase. Metabolic acidosis stimulates PEPCK and gluconeogenesis in the kidney [
29,
30]. These reports sport our hypothesis that the inhibition of H
+-ATPase reduces renal gluconeogenesis and plasma glucose level under starvation in diabetic rats. Acidosis and starvation enhances renal gluconeogenesis enzymes but not hepatic gluconeogenesis. The later is mainly regulated by insulin, glucagon and cortisol [
1‐
3,
29,
30]. Thus, H
+-ATPase blockade by BFM significantly reduced renal PEPCK, but not hepatic PEPCK expression.
In the present study, BFM showed antidiabetic effects in insulin-depleted STZ diabetic rats without significant changes in the body weight or blood pressure. As vacuolar H
+-ATPase is also expressed in the various cells including osteoclast, lung, testis and neuroendocrine cells [
12,
14], thus, it is possible that antidiabetic effects of BFM could be dependent upon the blocking effect on the non-renal cells. Recently it has been reported that adult mice with the conditional ablation of
Atp6ap demonstrated a significant reduction of plasma glucose, however, they also showed abnormalities in the intestine and hematopoietic cells [
31]. The limitation of this study is that one STZ rat treated with 200 nmol/kg body weight of BFM died during 24-h fasting because of hypoglycemia. Prof. Omura reported that all the mice survived with 0.6 mg/kg of setamycin, whereas more than 1.25 mg/kg of setamycin killed them [
15]. The reduction of food intake could be related to toxicity of BFM, and further studies are necessary to clarify the safety of BFM with longer periods of treatment.
Conclusion
The suppression of H+-ATPase by bafilomycin reduced renal gluconeogenesis and SGLT2 expression in the proximal tubules and decreased the plasma glucose level under starvation condition in diabetic rats.