Transamination is required for {alpha}-ketoisocaproate but not leucine to stimulate insulin secretion

Abstract

It remains unclear how α-ketoisocaproate (KIC) and leucine are metabolized to stimulate insulin secretion. Mitochondrial BCATm (branched-chain aminotransferase) catalyzes reversible transamination of leucine and α-ketoglutarate to KIC and glutamate, the first step of leucine catabolism. We investigated the biochemical mechanisms of KIC and leucine-stimulated insulin secretion (KICSIS and LSIS, respectively) using BCATm(-/-) mice. In static incubation, BCATm disruption abolished insulin secretion by KIC, D,L-α-keto-β-methylvalerate, and α-ketocaproate without altering stimulation by glucose, leucine, or α-ketoglutarate. Similarly, during pancreas perfusions in BCATm(-/-) mice, glucose and arginine stimulated insulin release, whereas KICSIS was largely abolished. During islet perifusions, KIC and 2 mM glutamine caused robust dose-dependent insulin secretion in BCATm(+/+) not BCATm(-/-) islets, whereas LSIS was unaffected. Consistently, in contrast to BCATm(+/+) islets, the increases of the ATP concentration and NADPH/NADP(+) ratio in response to KIC were largely blunted in BCATm(-/-) islets. Compared with nontreated islets, the combination of KIC/glutamine (10/2 mM) did not influence α-ketoglutarate concentrations but caused 120 and 33% increases in malate in BCATm(+/+) and BCATm(-/-) islets, respectively. Although leucine oxidation and KIC transamination were blocked in BCATm(-/-) islets, KIC oxidation was unaltered. These data indicate that KICSIS requires transamination of KIC and glutamate to leucine and α-ketoglutarate, respectively. LSIS does not require leucine catabolism and may be through leucine activation of glutamate dehydrogenase. Thus, KICSIS and LSIS occur by enhancing the metabolism of glutamine/glutamate to α-ketoglutarate, which, in turn, is metabolized to produce the intracellular signals such as ATP and NADPH for insulin secretion.

FIGURE 1.

Effects of BCATm disruption on nutrient-stimulated insulin secretion. A, islets isolated from BCATm^−/− and BCATm^+/+ mice were cultured overnight in PRMI 1640 supplemented with 10 mm glucose/2 mm glutamine. After preincubation in basal KRBH for 60 min, size-matched islets were pipetted into a 24-well Petri dish for a 60-min incubation with 2 ml of KRBH buffer containing 16.7 mm glucose, 10 mm leucine/2 mm glutamine, 10 mm KIC/2 mm Gln, and 10 mm dimethyl α-KG (DMα-KG)/2 mm Gln within a 37 °C 5% CO₂ incubator. Subsequently, the incubation buffer was collected for insulin immunoassay (n = 8 from two independent experiments). B, a second study was performed using the same condition as in A except stimulating with branched-chain keto acids, 10 mm KIC, 20 mm KMV, 10 mm KC, and 10 mm KIV all in the presence of 2 mm Gln (n = 8–10 from two independent experiments). Values are mean ± S.E.; a indicates p < 0.05 versus nontreated group of the same genotype; b indicates p < 0.05 versus BCATm^+/+ mice of the same treatment, and c indicates p < 0.05 versus BCATm^+/+ mice of KIC treatment.

FIGURE 2.

Effects of BCATm disruption on KICSIS during islet perifusion and pancreas perfusion. A, islets isolated from BCATm^−/− and BCATm^+/+ mice were cultured for 3 days in RPMI 1640with 10 mm glucose/2 mm glutamine. 100 islets were loaded into a perifusion chamber and immediately perifused with KRBH buffer added with 0.25% BSA. Islets were perifused with 2 mm glutamine from 0–120 min and a ramp of KIC from 0–25 mm at 50–100 min and 30 mm KCl at 120–130 min. Samples were collected every minute for insulin assay. Values are mean ± S.E. (n = 3). B, after a surgical procedure as described under “Materials and Methods,” the pancreas was perfused with oxygenated KRBH containing 1% BSA and 3% Dextran T70 through a catheter placed in the aorta. The effluent was collected at 1-min intervals from a cannula placed in the portal vein for insulin assay. The 90-min-long perfusion consisted of 2.8 mm glucose (G), 10 mm KIC, 2.8 mm glucose, 10 mm KIC/2 mm glutamine, 2.8 mm glucose, and 30 mm KCl from 0–20, 21–35, 36–50, 51–65, 66–80, and 81–90 min, respectively. Values are mean ± S.E. (n = 4 for BCATm^+/+ mice and n = 3 for BCATm^−/− mice).

FIGURE 3.

Effects of BCATm disruption on glucose-stimulated insulin secretion during pancreas perfusion and LSIS during islet perifusion. A, the pancreas was perfused with glucose (G) and arginine. The 95-min-long perfusion consisted of 2.8 mm glucose, 16.7 mm glucose, 2.8 mm glucose, 20 mm Arg·HCl, 2.8 mm glucose, and 16.7 mm glucose/Arg·HCl from 0–20, 21–40, 41–55, 56–70, 71–85, and 85–95 min, respectively. B, 100 islets were cultured in RPMI 1640 for 3 days and then loaded into a perifusion chamber perifused with KRBH buffer added with 0.25% BSA. Islets were perifused with 2 mm glutamine from 0–120 min and a ramp of leucine from 0–25 mm over 50–100 min followed by 30 mm KCl at 120–130 min (not shown). Samples were collected every minute for insulin assay. Values are mean ± S.E. (n = 3 for A and B).

FIGURE 4.

Effects of BCATm disruption on oxidation of KIC, leucine, and glucose as well as KIC conversion to amino acids. Groups of 25 islets placed into microfuge tubes housed within sealed 20-ml scintillation vials were incubated with [U-¹⁴C]KIC, [U-¹⁴C]leucine, and [U-¹⁴C]glucose to measure KIC (A), leucine (C), or glucose oxidation (D) as described under “Materials and Methods.” The conversion of KIC into amino acids (AA; B) was evaluated in the same experiment of ¹⁴CO₂ production from [U-¹⁴C]KIC. Three or four independent experiments were performed. Values are mean ± S.E. *, p < 0.05; n = 8–16.

FIGURE 5.

Effects of BCATm disruption on islet contents of ATP, malate, and α-KG as well as the NADP/NAD ratio. A, After an overnight culture, five islets per condition were transferred into a 1.5-ml microfuge tube and incubated with 0.6 ml KRBH/0.5% BSA without or with stimulators (16.7 mm glucose, 10 mm leucine/2 mm glutamine), 10 mm KIC/2 mm Gln, or 10 mm dimethyl α-KG (DMα-KG)/2 mm Gln at 37 °C for 60 min. ATP was measured using a Bioluminescent Assay kit as described under “Materials and Methods.” B and C, ∼200 overnight-cultured islets for each treatment were preincubated in KRBH/0.5% BSA for 60 min and then incubated in a 1.5-ml microfuge tube stimulated with 16.7 mm glucose, 10 mm KIC/2 mm Gln, or basal buffer for 60 min. Malate was measured using an enzymatic analysis coupled with fluorometry, and α-KG was measured using more sensitive indirect fluorometry. D, ∼200 overnight-cultured islets for each treatment were preincubated in KRBH/0.5% BSA for 60 min and then incubated in a 1.5-ml microfuge tube stimulated with 10 mm KIC/2 mm Gln or basal buffer for 60 min. NADPH/NADP⁺ ratios were measured using an enzymatic cycling assay coupled with fluorometry. Values are mean ± S.E. a, p < 0.05 versus nontreated group of the same genotype; b, p < 0.05 versus BCATm^+/+ mice of the same treatment (n = 10 from two independent experiments for ATP assay; n = 4–5 for malate and α-KG assays; and n = 5 for the NADPH/NADP⁺ ratio measurement.

FIGURE 6.

Effects of BCATm disruption on food intake, plasma insulin, and glucose concentrations in response to fasted refeeding. Female mice were fasted for 21 h and then refed with diet choice of normal chow/amino acid purified branched-chain amino acid-free diet for 6 h. Body weight (B.W.)-normalized food intake during the 6-h refeeding period was recorded (A), and blood samples were collected at 0, 1, 3, 4.5, and 6 h of refeeding for measuring plasma insulin (B) and glucose (C). Values are mean ± S.E. *, p < 0.05 versus BCATm^+/+ mice at the same time point; n = 13 for BCATm^+/+ and 12 for BCATm^−/− mice.