Disordered branched chain amino acid catabolism in pancreatic islets is associated with postprandial hypersecretion of glucagon in diabetic mice

 Dysregulation of glucagon is associated with the pathophysiology of type 2 diabetes

• Here, we investigated the effects of nutrients on glucagon secretion using islets isolated from diabetic mice and palmitate-treated islets.
• In addition, we analyzed the expression levels of branched chain amino acid (BCAA) catabolism-related enzymes and their metabolites in diabetic islets and found that protein, but not carbohydrate or lipid, increased plasma glucagon levels.

Abbreviations

• BCAA branched chain amino acid
• Aminotransferase 2
• Keto acid dehydrogenase kinase
• α-keto acid oxidase enzyme
• L-amino acid transporter 4F2hc
• Cell-surface antigen heavy chain
• BCA-CoA
• A.k.a. acyl-CAA
• Protein phosphatase, Mg2+/Mn2+ dependent 1K
• DNA methyltransferase

Dysregulation of glucagon is associated with the pathophysiology of type 2 diabetes

• Glucagon catabolizes hepatic amino acids, decreasing serum amino acid levels
• Recently, decreased serum amino acids were reported to reduce mechanistic target of rapamycin (mTOR) signaling in α cells and suppress α cell proliferation
• A mutual link clearly exists between glucagon and amino acids under physiological conditions
• However, the pathological significance of nutrient-associated glucagon secretion is unclear
• One critical reason for these controversies is the unreliable measurement of plasma glucagon levels.
• Because recently more accurate glucagon sandwich ELISA has been developed, in this study, we assessed the effects of various nutrients on plasma glucose levels in mice by using this new system
• We found that protein, rather than glucose or lipid, increases plasma glucoseagon levels more markedly in diabetic mice than in healthy controls
• BCAAs, including leucine, valine, and isoleucine are essential amino acids

Animal studies

• Male C57BL/6J mice were randomly assigned to a normal chow diet (CE-2) or a 60% high fat diet (HFD; 60.7% fat, 17.9% protein and 21.4% carbohydrate, Oriental Yeast, Tokyo, Japan) and given 50 mg/kg streptozotocin (STZ) intraperitoneally.
• For the glucose tolerance test, mice were orally administered glucose (1.5 g/kg body weight) after 16 h fasting and injected with 0.75 U/kg insulin and blood glucose levels measured with a glucometer.

Single nutrient loading test

• Mice were orally administered whey protein, intralipos, or glucose after 16 h fasting, with every nutrient adjusted to 1.0 kcal/mL and given as 6 μL/g body weight.
• For the amino acid loading test, mice were orally or intraperitoneally administered a BCAA mixture containing valine, leucine, and isoleucine. The component ratio was adjusted to that of whey Protein, and the other amino acid mixtures were prepared as essential amino acids excluding BCAAs, and nonessential amino acids (NEAAs).

Glucagon secretion from isolated islets.

• Islets were isolated from db/db, HFD-fed, and control mice by collagenase digestion and subsequent centrifugation as described previously [29].
• Size matched islets were handpicked and incubated in 700 μL of HEPES-buffered Krebs-Ringer bicarbonate buffer (HKRB) containing 5 mM glucose and 2 mM glutamine with or without 40 mM BCAAs (Leu:Ile:Val = 2:1:1) for 1 h at 37°C.

Quantitative RT-PCR

• Total RNAs isolated from mouse islets, mouse kidney, αTC1.6 cells, and MIN6 cells were reverse-transcribed to cDNA.
• The cDNA (1 μg) and the target mRNA expression levels were evaluated relative to the GAPDH or β-actin mRNA levels.

Immunoblot analysis

• Total cell lysates were prepared by polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes.

Calcium imaging:

• To specifically label pancreatic α cells with red fluorescence, glucagon-Cre mice were crossed with tdTomato reporter mice.

Amino acid and BCAA metabolite analyses

• To extract amino acids, mouse plasma or isolated islets were plunged into 80% methanol containing 10 µM 2-Morpholinoethanesulfonic acid as an internal standard, and homogenized.
• The solution was centrifuged at 12,000 × g for 10 min at 4°C, and the supernatant was filtered through a Captiva ND Lipids to remove proteins and phospholipids before being dried up and resuspended in water.

Statistical Analysis

• Data are presented as the mean ± SE and analyzed by the paired t-test, Student's or Welch's T-test as appropriate.

Postprandial hyperglucagonemia was observed in various diabetic mouse models.

• The body weights of db/db, HFD+STZ2, and HFD-fed mice were significantly higher than those of the control mice, whereas HFD/STZ5 mice’s body weights were comparable to the control (Fig. 1).
• All four diabetic models had significantly higher plasma glucagon levels in the fed state than the control model.

Protein increased plasma glucagon levels in various diabetic mouse models

• To confirm the influence of restraint stress on the parameters examined in this study, we conducted a water tolerance test, in which we orally administered the same volume of water as the nutrients to the diabetic mice and evaluated plasma glucose, insulin, and blood glucose levels.
• Water administration did not increase plasma glucose or insulin levels, but it significantly increased blood glucose in HFD+STZ2 and HFD-fed mice.

BCAAs increased plasma glucagon levels in diabetic mice.

• Of the three types of amino acids examined, BCAA increased plasma glucose levels the most in HFD-fed mice, followed by EAAs and NEAAs
• Blood glucose levels were increased by only BCA as and not in the other diabetic mouse models, probably due to the parallel increase in both glucagon and insulin

BCAAs directly stimulated glucagon secretion in control islets, which was further enhanced in diabetic islets.

• In the previous studies using oral administration, we could not rule out the secondary effects of incretin secreted from the gastrointestinal tract on glucagon, but this was not confirmed by the direct mechanism in pancreatic α cells (Supplemental Fig. 7).
• We examined the effects of intraperitoneally injected BCA as on plasma glucagon levels in db/db mice, and found that the effects were mediated by intracellular transport of BCAA as well.

BCAA catabolism disorder in db/db islets was associated with higher glucagon secretion

• Plasma BCAA levels have been reported to be increased in obese men and type 2 diabetes mellitus patients compared to healthy subjects, and in diabetic islets, as well
• The expression levels of BCAA-related genes were also found to be significantly decreased in the islets compared to controls
• These results support the hypothesis that the BCAA pathway is disordered in diabetic islets
• Chemical BCKDK inhibitor BT2 canceled BCAA hypersecretion of glucagon in islets

Disordered BCAA catabolism in palmitate-treated islets was associated with enhanced BCAA-induced glucagon secretion

• Pancreatic β cell dysfunction was reported to be caused by lipotoxicity [37]
• Insulin secretion was impaired by incubation with Palmitate in isolated islets [38].
• To test this, we treated islets with 0.5 mM palmitamate for 24 h and measured the mRNA expression of BCAA Catabolism-related enzymes.
• Although the expression levels of Bcat2 and Ppm1k were unchanged after the islet treatment, we measured a significant decrease in Bckdha and an increase in BCckdk in the islets (Fig. 6A-D).

Discussion

• Hyperglucagonemia is more apparent in the fed state than the fasted state in various diabetic mouse models.
• These results suggest that BCAA-induced hypersecretion of glucagon in diabetic mice is independent of insulin or blood glucose levels and due, instead, to the intrinsic mechanism in α cells.
• Because enhanced glucagon secretion after a meal might exacerbate postprandial hyperglycemia, these results have important clinical implications for diet therapy in diabetes.

Conclusion

• Disordered BCAA catabolism in pancreatic islet cells is associated with postprandial hypersecretion of glucagon in diabetic mice.

• Arg-containing proteins are digested, as all other proteins, by an array of secreted enzymes from stomach, pancreas, and small intestine, and by aminopeptidases at the brush border membrane. The combined action of these enzymes releases small peptides and free amino acids.
• Di- and tripeptides are taken up through the hydrogen ion/peptide cotransporters 1 and 2 (PepT1, SLC15A1).
• Sodium-dependent transport systems are known to facilitate Arg uptake into enterocytes of the distal small intestine.

Transport and cellular uptake

• Blood circulation: Normal plasma concentrations of Phe are around 50 μmol/l (Artuch et al., 1999). Concentrations are not significantly influenced by Phe intake.
• The amino acid enters cells from blood via several transporters, including system 1 (TAT1) and LAT1, whose expression patterns vary considerably between specific tissues. LAT1 also transports D-phenylalanine.

Blue Diaper Syndrome

• It is a rare familial disease in which hypercalcemia and nephrocalcinosis are associated with a defect in the intestinal transport of tryptophan.
• The clinical course is characterized by failure to thrive, recurrent unexplained fever, infections, marked irritability, and constipation.- Treatment consists of glucocorticoids and a low-calcium, low-vitamin D diet.

System L

• This is the major Na+-independent system in the intestinal basolateral membrane for the transport of neutral amino acids.
• Recent studies on the molecular aspects of this transport system have identified two different isoforms of system L belonging to the SLC7 transporter gene family. Both of them require 4F2hc, also known as CD98, as a common subunit.

System L

• A Na+-independent transporter of essential amino acids, including l-leucine and l-phenylalanine, found in both the MVM and BM of the ST, exchanging nonessential amino acids for predominantly essential acids.
• This transporter is a homodimer, made up of a light chain protein, L-type Amino acid Transporter, LAT1 (SLC7A5), covalently linked to a heavy chain transmembrane protein, 4F2hc/CD98, which increases in the placenta from mid-gestation to term.

Large neutral amino acid transporters (System L)

• These are involved in the transport of large neutral, branched or aromatic amino acids including essential amino acids and several amino-acid-related compounds, such as L-dopa, α-methyldopa and gabapentin, across blood tissue barriers and epithelia.
• Both LAT1 and LAT2 transport amino acids in a Na+-independent manner, however, LAT1 has high affinity while LAT2 has low affinity.

Transport and Cellular Uptake

• Blood circulation: Normal plasma concentrations of Tyr are around 66 µmol/l (Artuch et al., 1999).
• The amino acid enters cells from the blood via several transporters, including system T (TAT1) and LAT1, whose expression patterns vary considerably between specific tissues.
• Materno-fetal transfer: The exchanger LAT1 appears to be the major route for Tyr traveling from maternal blood into the syncytiotrophoblast (Ritchie and Taylor, 2001).

Transport and cellular uptake

• Blood circulation: The plasma concentration of Trp (typically around 50 μmol/l) decreases in response to low dietary intake
• Uptake from blood into uses various transporters, including system T (TAT1), LAT1, and LAT2, whose expression patterns vary considerably between specific tissues and the Blood-brain barrier
• Trp competes with branched-chain amino acids (valine, leucine, isoleucine) and other large neutral amino acids for the transport into brain.

Transport and cellular uptake

• Blood circulation: While most Lys in blood is part of proteins, the concentration of free Lys in plasma is around 195 μmol/l.
• Materno-fetal transfer: Since Lys is an essential amino acid, the fetus is fully dependent on transfer across the placenta. Several members of system y+ mediate uptake from maternal circulation into the syntrophoblast layer.
• Blood-brain barrier: The 4F2-anchored exchange complex y+LAT2 is known to contribute significantly.

Reference

• 10.1016/j.jnutbio.2021.108811