Diabetes Metab J.  2023 Sep;47(5):653-667. 10.4093/dmj.2022.0244.

CycloZ Improves Hyperglycemia and Lipid Metabolism by Modulating Lysine Acetylation in KK-Ay Mice

• Jeon J
• Lee D
• Kim B
• Park BY
• Oh CJ
• Kim MJ
• Jeon JH
• Lee IK
• Park O
• Baek S
• Lim CW
• Ryu D
• Fang S
• Auwerx J
• Kim KT
• Jung HY

• KMID: 2546124
• DOI: http://doi.org/10.4093/dmj.2022.0244

Abstract

Background
CycloZ, a combination of cyclo-His-Pro and zinc, has anti-diabetic activity. However, its exact mode of action remains to be elucidated.
Methods
KK-Ay mice, a type 2 diabetes mellitus (T2DM) model, were administered CycloZ either as a preventive intervention, or as a therapy. Glycemic control was evaluated using the oral glucose tolerance test (OGTT), and glycosylated hemoglobin (HbA1c) levels. Liver and visceral adipose tissues (VATs) were used for histological evaluation, gene expression analysis, and protein expression analysis.
Results
CycloZ administration improved glycemic control in KK-Ay mice in both prophylactic and therapeutic studies. Lysine acetylation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha, liver kinase B1, and nuclear factor-κB p65 was decreased in the liver and VATs in CycloZ-treated mice. In addition, CycloZ treatment improved mitochondrial function, lipid oxidation, and inflammation in the liver and VATs of mice. CycloZ treatment also increased the level of β-nicotinamide adenine dinucleotide (NAD⁺), which affected the activity of deacetylases, such as sirtuin 1 (Sirt1).
Conclusion
Our findings suggest that the beneficial effects of CycloZ on diabetes and obesity occur through increased NAD⁺ synthesis, which modulates Sirt1 deacetylase activity in the liver and VATs. Given that the mode of action of an NAD+ booster or Sirt1 deacetylase activator is different from that of traditional T2DM drugs, CycloZ would be considered a novel therapeutic option for the treatment of T2DM.

Keyword

Acetylation; Diabetes mellitus, type 2; NAD; Obesity

Figure

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  Fig. 1. CycloZ administration ameliorates type 2 diabetes mellitus and obese phenotypes in KK-Ay mice. (A) Body weight changes during 20 weeks of administration (n=7–8). (B) Weight of each organ after sacrifice (n=7–8). (C) Oral glucose tolerance test for 2 hours after 16 hours fasting and glucose administration (2 g/kg) at 10 weeks of treatment (n=14–16). (D) Blood glucose level after 16 hours fasting at 10 weeks of treatment (n=14–16). (E) Glycosylated hemoglobin (HbA1c) levels in KK-Ay mice at 11 weeks of treatment (n=7–8). (F) Plasma insulin concentration (n=14–16). (G) Plasma free fatty acid concentration (n=7–8). (H) Plasma triglyceride concentration (n=7–8). (I) Plasma high-density lipoprotein (HDL) concentration (n=7–8). (J) Plasma adiponectin level was measured by Western blot (n=7–8). (K) mRNAs expression related to fatty acid and cholesterol synthesis in liver (n=7–8). (L, M) H&E staining of liver and epididymal adipose tissue (EAT). Data shown represent mean±standard error of the mean. Unpaired Student’s t-tests. CTRL, control; MAT, mesenteric adipose tissue; SAT, subcutaneous adipose tissue; BAT, brown adipose tissue; Srebf, sterol regulatory element-binding transcription factor; Srebp, sterol regulatory-element binding protein; Fasn, fatty acid synthase; Hmgcr, 3-hydroxy-3-methylglutaryl-CoA reductase. aP≤0.05, bP≤0.01, cP≤0.001, dP≤0.0001.

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  Fig. 2. CycloZ recovers inflammation and immune cell infiltration in liver and visceral adipose tissues of KK-Ay mice. (A) mRNAs expression level related inflammatory cytokines and infiltrated monocyte in liver and mesenteric adipose tissue (n=7–8). (B, C) Tumor necrosis factor α (TNFα) and macrophage chemoattractant protein 1 (MCP-1) protein levels in liver (B) and epididymal adipose tissue (EAT) (C) (n=7–8). (D, E) Expression of TNFα, MCP-1, F4/80, and CD11b in liver (D) and EAT (E) was measured by immunohistochemistry. Data shown represent mean±standard error of the mean. Unpaired Student’s t-tests. CTRL, control; Il-1b, interleukin 1 beta; WAT, white aidpose tissue. aP≤0.05, bP≤0.01, cP≤0.0001.

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  Fig. 3. CycloZ improves mitochondrial function via sirtuin 1 deacetylase. (A) Levels of acetylated lysine on p65 in liver and white adipose tissue (WAT) (n=14–16). (B) Acetylated lysine levels on peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in liver and epididymal adipose tissue (EAT) (n=14–16). (C) Acetylated lysine levels on liver kinase B1 (LKB1) in liver and EAT (n=14–16). (D) Phosphorylated AMP-activated kinase (AMPK) (T172) in liver and EAT (n=7–8). (E) mRNAs expression level related to mitochondrial biogenesis in liver and mesenteric adipose tissue (MAT) (n=7–8). (F) mRNAs expression level related to lipid oxidation in liver and MAT (n=7–8). (G) Oxygen consumption rate (OCR) was measured in alpha mouse liver 12 (AML12) after 16-hour treatment of CycloZ with and without palmitate. (H) MitoTracker staining for measuring mitochondrial mass. Data shown represent mean±standard error of the mean. Unpaired Student’s t-tests. CTRL, control; IP, immunoprecipitation; Ac-K, acetylated-lysine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Foxo1, forkhead box O1; Esrra, estrogen related receptor alpha; Nrf1, nuclear respiratory factor 1; Tfam, transcription factor A, mitochondrial; Ucp1, uncoupling protein 1; Ppara, peroxisome proliferator-activated receptor alpha; Cpt1a, carnitine palmitoyltransferase 1A; Ppargc1a, PPARG coactivator 1 alpha; CCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rot/AA, rotenone/antimycin A; BSA, bovine serum albumin; PAL, palmitic acid. aP≤0.05, bP≤0.01, cP≤0.001, dP≤0.0001.

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  Fig. 4. CycloZ administration increases the level of β-nicotinamide adenine dinucleotide (NAD+) content by modulating the expression of genes involved in NAD+ synthesis. (A) NAD+/nicotinamide adenine dinucleotide (NADH) ratio in liver and epididymal adipose tissue (EAT) (n=7–8). (B) Quantification of NAD+ in liver and EAT. (n=7–8). (C, D) mRNAs expression levels related to NAD+ synthesis in liver (C) and mesenteric adipose tissue (D) (n=7–8). Data shown represent mean±standard error of the mean. Unpaired Student’s t-tests. CTRL, control; WAT, white adipose tissue; Naprt, nicotinate phosphoribosyltransferase; Nmnat, nicotinamide mononucleotide adenyltransferase; Nads, NAD synthase; Nampt, nicotinamide phosphoribosyltransferase; Nrk, nicotinamide riboside kinase; Pnp, purine nucleoside phosphorylase; Tdo, tryptophan 2,3-dioxygenase; Qprt, quinolinate phosphoribosyltransferase; Acmsd, aminocarboxymuconate semialdehyde decarboxylase; Nqo1, NAD(P)H quinone dehydrogenase 1. aP≤0.05, bP≤0.01, cP≤0.001.

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  Fig. 5. CycloZ administration improves glucose control in a model of severe established type 2 diabetes mellitus. (A) Oral glucose tolerance test for 2 hours after 16 hours fasting and glucose administration (2 g/kg) at 10 weeks of treatment (n=8–10). (B) Glycosylated hemoglobin (HbA1c) level at 11 weeks of treatment (n=8–10). (C) Acetylated lysine levels on peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and liver kinase B1 (LKB1) in liver (n=8–10). (D) Phosphorylated AMP-activated kinase (AMPK) (T172) in liver (n=8–10). (E) Increased the level of β-nicotinamide adenine dinucleotide (NAD+)/nicotinamide adenine dinucleotide (NADH) ratio and quantification of NAD+ in liver (n=8–10). (F) NAD+ synthesis related mRNAs expression levels in liver (n=8–10). Data shown represent mean±standard error of the mean. Unpaired Student’s t-tests. CTRL, control; IP, immunoprecipitation; IB, iimmunoblotting; Ac-K, acetylated-Lysine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Naprt, nicotinate phosphoribosyltransferase; Nmnat, nicotinate/nicotinamide mononucleotide adenyltransferase; Nads, NAD synthase; Nampt, nicotinamide phosphoribosyltransferase; Nrk, Nik related kinase; Pnp, purine nucleoside phosphorylase; Tdo, tryptophan 2,3-dioxygenase; Qprt, quinolinate phosphoribosyltransferase; Nqo1, NAD(P)H quinone dehydrogenase 1. aP≤0.05, bP≤0.01, cP≤0.001, dP≤0.0001.

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