Chronic oscillating glucose challenges disarrange innate immune homeostasis to potentiate the variation of neutrophil–lymphocyte ratio in rats with or without hidden diabetes mellitus

Figures & data

Table 1 Kidney weight, kidney index, heart weight, heart index, blood glucose, water intake, food intake, urine output and 24 h urinary albumin excretion in sham and HD groups on experimental day P27

Variable	Sham (n=8)	HD (n=7)
Kidney weight (g)	1.46±0.03	1.85±0.13*
Kidney index	6.91±0.20	9.07±0.61*
Heart weight (g)	0.66±0.02	0.62±0.01
Heart index	3.10±0.10	3.06±0.16
Blood glucose (mg/dL)	75±2	83±5
Water intake (mL/day)	28±3	158±42*
Food intake (g/day)	15±1	33±7*
Urine volume (mL/day)	9±1	138±43*
Urinary albumin (μg/day)	18.96±14.52	83.09±56.23*

Notes: Values are expressed as mean±SEM. Kidney index: ratio of kidney weight/body weight (mg/g); heart index: ratio of heart weight/body weight (mg/g).

* p<0.05 sham vs HD.

Abbreviations: HD, hidden diabetes; P27, posttreatment day 27.

Figure 1 Alloxan produced the development of HD in female rats (sham n=8, HD n=7).

Notes: (A) FBG levels during experimental period P0–P27. (B) OGTT after 7 days of alloxan treatment. (C) OGTT after 27 days of alloxan treatment. (D) OGTT AUC after 7 and 27 days of alloxan treatment. (E) Body weights were measured daily during experimental period P0–P27. (F) Body weight changes during experimental period P1–P27. The changes were obtained from the difference in body weight between posttreatment day P1–P27 and pretreatment day P0. The data for OGTT AUC were analyzed by unpaired Student’s t-test. The data for FBG, OGTT, body weight, and body weight changes were analyzed by 2-way analysis of variance with Tukey tests for posthoc comparisons. *p<0.05 and ***p<0.001 sham vs HD; ⁺⁺⁺p<0.001 P7 HD vs P27 HD.
Abbreviations: HD, hidden diabetes; FBG, fasting blood glucose; OGTT, oral glucose tolerance test; AUC, area under the curve; P0, prealloxan treatment day; P1, posttreatment day 1; P7, posttreatment day 7; P27, posttreatment day 27.

Table 2 Leukogram of sham and HD groups on experimental days P7 and P27

Variable	P7		P27
	Sham (n=6)	HD (n=5)	Sham (n=8)	HD (n=7)
Leukocytes (×10^9/L)	12.38±1.52	11.70±0.85	13.70±1.56	11.36±1.49
Neutrophils (%)	12.79±1.44	25.39±2.40*	15.08±2.46	26.68±2.82*
Lymphocytes (%)	83.36±1.43	67.83±2.37*	76.01±2.63	65.78±3.12*
Monocytes (%)	2.55±0.73	6.52±1.57	5.63±1.11	6.73±2.29
Eosinophils (%)	1.11±0.25	0.26±0.13	0.59±0.30	0.54±0.32
Basophils (%)	0.17±0.12	0.00±0.00	0.00±0.00	0.00±0.00

Notes: Values are expressed as mean±SEM.

* p<0.05 sham vs HD.

Abbreviations: HD, hidden diabetes; P7, posttreatment day 7; P27, posttreatment day 27.

Figure 2 Leukogram of female rats with or without alloxan treatment on P7 (n=5 and n=6, respectively) and P27 (n=7 and n=8, respectively).

Notes: (A) Leukocyte counts after 7 or 27 days of alloxan treatment. (B) Percentage neutrophils after 7 or 27 days of alloxan treatment. (C) Percentage lymphocytes after 7 or 27 days of alloxan treatment. (D) NLR resulting from (B) and (C). (E) Correlation between percentage neutrophils and NLR on P27. (F) Correlation between percentage lymphocytes and NLR on P27. (G) Percentage neutrophils in sham, HD, and DM groups on P27. (H) Percentage lymphocytes in sham, HD, and DM groups on P27. (I) NLR resulting from (G) and (H). The data from the leukogram were analyzed by analysis of variance with further tests for posthoc comparisons. Relationships between parameters were compared using Pearson correlation analysis. *p<0.05 and **p<0.01 sham vs HD.
Abbreviations: P7, posttreatment day 7; P27, posttreatment day 27; NLR, neutrophil–lymphocyte ratio; HD, hidden diabetes; DM, diabetes mellitus.

Figure 3 Leukogram of female rats with or without OG and OG+GSH treatments on P7 (n=7 for all groups).

Notes: (A) Leukocyte counts in PC or P1h treatment. (B) Lymphocyte number after PC or P1h treatment. (C) Percentage lymphocytes after PC or P1h treatment. (D) Neutrophil number in PC or P1h treatment. (E) Percentage neutrophils in PC or P1h treatment. (F) NLR of PC or P1h treatment. (G) Correlation between changes in blood glucose and changes in lymphocyte number. The data from the leukogram were analyzed by 2-way analysis of variance with Tukey tests for posthoc comparisons. Relationships between parameters were compared using Pearson correlation analysis. *p<0.05, **p<0.01, and ***p<0.001 PC vs P1h.
Abbreviations: OG, oscillating glucose; GSH, glutathione; P7, posttreatment day 7; PC, precontrol; P1h, 1 h post-glucose; NLR, neutrophil–lymphocyte ratio.

Figure 4 Development of pancreatic and renal pathology of HD with diabetes mellitus on days P7 and P27 after alloxan treatment.

Notes: (A) Islet area (P7: both sham and HD n=18; P27: sham n=27, HD n=21). (B) β-cells/islet area ratio (P7: sham n=173, HD n=84; P27: sham n=135, HD n=58). (C) Insulin+β-cells and glucagon+α-cells in sham group on P27 (magnification ×400). (D) Insulin+β-cells and glucagon+α-cells in HD group on P27 (magnification ×400). (E) Glomerular area (P27: sham n=1076, HD n=812). (F) Glomerular volume (P27: sham n=1076, HD n=812). ***p<0.001 sham vs HD.
Abbreviations: HD, hidden diabetes; P7, posttreatment day 7; P27, posttreatment day 27.

Figure 5 Relationships among leukogram data, glucose metabolic changes, and kidney index values after 27 days of alloxan treatment.

Notes: OGTT AUC values were positively correlated with the percentage of neutrophils (A) and NLR (B). Kidney index values (kidney weight/body weight ratio) were also positively correlated with the percentage neutrophils (C) and NLR (D). The percentage of lymphocytes was negatively correlated with OGTT AUC (E) and kidney index (F). The kidney index values were positively correlated with OGTT AUC (G). Relationships between parameters were compared using Pearson correlation analysis.
Abbreviations: OGTT, oral glucose tolerance test; AUC, area under the curve; NLR, neutrophil–lymphocyte ratio; KW/BW, kidney weight/body weight ratio.