1. Arachidonic acid and lipoxinA4 attenuate streptozotocin-induced cytotoxicity to RIN5 F cells in vitro and type 1 and type 2 diabetes mellitus in vivo 
Naveen K.V. Gundala, M.Sc., Vegi G.M. Naidu, Ph.D., Undurti N. Das, M.D. 
Nutrition 
Volume 35, Pages (March 2017)
DOI: /j.nut
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Fig. 1
Dose optimization studies with streptozotocin (STZ), arachidonic acid (AA), and lipoxin A4 (LXA4) on RIN5 F cell viability in vitro. (A) RIN5 F cells were treated with various doses (3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 mM) of STZ for 8, 12, 16, 20, 24, and 28 h in vitro. (B) Dose and time optimization study with AA on RIN5 F cells. (C and D) RIN5 F cells were supplemented with 1, 5, 10, and 50 ng/mL of LXA4 for 0.5, 1, 2, 4, 6, 12, 24, and 48 h. All the above set of experiments were done in triplicate and repeated on more than three to six separate occasions, and values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control.
Nutrition  , 61-80DOI: ( /j.nut )
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Fig. 2
Effect of pre- and simultaneous-treatment schedules with ω-6 (AA and GLA), n-3 (EPA and DHA) PUFAs on STZ-induced cytotoxicity to RIN5 F cells. (A and B) Doses of 5, 10, and 15 μg/mL n-3 PUFAs (DHA and EPA) were used in pre- and simultaneous-treatment schedules, and a dose of 21 mM of STZ for 24 h was used to study their (PUFAs) effects on RIN5 F cells. The pretreatment period is for 5 h. (C and D) Doses of 5, 10, and 15 μg/mL of n-6 PUFAs (AA and GLA) were used in pre- and simultaneous-treatment schedules, and a dose of 21 mM of STZ was used to study their (PUFA) effects on RIN5 F cells. All the above set of experiments was done in triplicate on two separate occasions (n = 6), and values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ. AA, arachidonic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; GLA, γ-linolenic acid; PUFA, polyunsaturated fatty acid; STZ, streptozotocin.
Nutrition  , 61-80DOI: ( /j.nut )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Fig. 3
Effect of pre- and simultaneous treatment with LXA4 on STZ induced cytotoxicity to RIN5 F cells in vitro. (A) RIN5 F cells were pretreated with 1, 5, 10, and 50 ng/mL of LXA4 to study its modulatory action on STZ-induced (21 mM) cytotoxic action. (B) RIN5 F cells were simultaneously treated with 1, 5, 10, and 50 ng/mL of LXA4 and STZ (21 mM) to evaluate the cytoprotective action of LXA4 in vitro. All the above set of experiments was done in triplicate on two separate occasions (n = 6), and values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ. LXA4, lipoxin A4; STZ, streptozotocin.
Nutrition  , 61-80DOI: ( /j.nut )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Fig. 4
Effect of STZ on LXA4 secretion by RIN5 F cells and its modulation by various PUFAs in vitro. (A) RIN5 F cells were treated with various doses (3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 mM) of STZ for 24 h. The LXA4 was estimated by ELISA in the supernatant of cultures. (B) RIN5 F cells were treated with 10 μg/mL PUFAs ± STZ (21 mM) for 24 h. The LXA4 was estimated in the supernatant of the cell cultures. All the above set of experiments was done in triplicate on two separate occasions (n = 6), and values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ. ELISA, enzyme-linked immunosorbent assay; LXA4, lipoxin A4; PUFA, polyunsaturated fatty acid; STZ, streptozotocin.
Nutrition  , 61-80DOI: ( /j.nut )
Copyright © 2016 Elsevier Inc. Terms and Conditions

6. Fig. 5
Assessment of DNA fragmentation and apoptosis/necrosis and expression of Pdx1, NF-κb, IKB, and β-actin genes: (A) Treated RIN5 F cells were harvested, and DNA was isolated and loaded in the submerged gel electrophoretic system in presence of ethidium bromide as a fluorescence dye. The bands were observed and their photographs taken by using gel documentation system. (B) STZ-treated RIN5 F cells were assessed for apoptosis and necrosis induction by STZ and its modulation by LXA4 using acridine orange-propidium iodide double-staining method. Cells that are stained green and fully intact are viable, those stained in green and shrunken in size are those showing apoptosis, and those stained red and showing features of disintegration are considered to have undergone necrosis. The number of cells showing features of apoptosis, necrosis, or normality was counted manually, and the values obtained were expressed as percentage of total number of cells counted. All values are expressed as mean ± SEM. aP ≤ 0.05 compared with the viable cells in the untreated control; bP ≤ 0.05 compared with the number of apoptotic cells in the untreated control; cP ≤ 0.05 compared with the number of necrotic cells in the untreated control; dP ≤ 0.05 compared with the number of viable cells following STZ treatment; eP ≤ 0.05 compared with the number of apoptotic cells following STZ treatment; fP ≤ 0.05 compared with the number of necrotic cells following STZ treatment. (C and D) Gene expression studies were performed in RIN5 F cells treated simultaneously with LXA4 and STZ. The percentage of change in the expression of genes: Pdx1, NF-κb, IKB, and β-actin were studied in RIN5 F cells treated simultaneously with LXA4 and STZ by using semiquantitative polymerase chain reaction method. The equality of sample loading was confirmed by beta actin gene expression. All the above set of experiments was done wherein each group consisted of n = 6, and values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ. LXA4, lipoxin A4; PUFA, polyunsaturated fatty acid; STZ, streptozotocin.
Nutrition  , 61-80DOI: ( /j.nut )
Copyright © 2016 Elsevier Inc. Terms and Conditions

7. Fig. 6
Effect of AA on STZ-induced T1DM in Wistar rats. (A) Experimental protocol: after 7 d of acclimatization, animals received 10 μg/mL of AA intraperitoneally (IP) or oral once every week, whereas STZ (45 mg/kg of body weight was given only on day 1. (B) Plasma blood glucose levels in animals: blood glucose estimation was performed once in 10 d until the end of the study. All values are expressed as mean ± SEM. aP ≤ 0.05 compared with 10th-day control values. bP ≤ 0.05 compared with 20th-day control values. cP ≤ 0.05 compared with 30th-day control values. dP ≤ 0.05 compared with plasma glucose levels seen on day 10 after STZ alone administration. eP ≤ 0.05 compared with plasma glucose levels seen day 20 after STZ administration. fP ≤ 0.05 compared with plasma glucose levels seen on day 30 after STZ administration. All the above set of experiments was done in triplicate on two separate occasions (n = 6) and values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ. (C) Body weight of Wistar rats treated with AA ± STZ. Body weights were measured once in 7 d until the end of the study. All the values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ control (positive control). (D) Measurement of LXA4 levels in plasma of AA ± STZ treated animals at the end of the study (day 30). (E) Plasma insulin levels in AA ± STZ treated Wistar rats. Insulin estimation was done in the plasma collected at the end of the study. All values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ control (positive control group). (F) Plasma TNF-α level in AA ± STZ treated rats. TNF-α measurement was done in plasma collected once in every 10 d until the end of the study. All values are expressed as mean ± SEM. aP ≤ 0.05 compared with the day 10 control; bP ≤ 0.05 compared with the day 20 control; cP ≤ 0.05 compared with the day 30 control; dP ≤ 0.05 compared to the day 10 STZ control; eP ≤ 0.05 compared with the day 20 STZ control; fP ≤ 0.05 compared with the day 30 STZ control. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ control. All the above studies were done wherein each group consisted of n = 6, and all values are expressed as mean ± SEM. AA, arachidonic acid; STZ, streptozotocin; T1DM, type 1 diabetes mellitus; TNF, tumor necrosis factor.
Nutrition  , 61-80DOI: ( /j.nut )
Copyright © 2016 Elsevier Inc. Terms and Conditions

8. Fig. 7
Effect of COX and LOX inhibitors on the beneficial action of AA against STZ-induced T1DM. (A) Experimental protocol: after 7 d of acclimatization, animals received both STZ 45 mg/kg body weight and AA 10 μg/mL intraperitoneally at the same time on day 1. AA was continued every day for 1 wk and later once a week until end of the study. 1 mM of COX and LOX inhibitors were given intraperitoneally only on day 1 along with STZ and AA. (B) Plasma blood glucose levels. Blood glucose estimation was performed once in 10 d until end of the study. All values are expressed as mean ± SEM. aP ≤ 0.05 compared with day 10 control values; bP ≤ 0.05 compared with day 20 control values; cP ≤ 0.05 compared with day 30 control values; dP ≤ 0.05 compared with day 10 STZ control values; eP ≤ 0.05 compared with day 20 STZ control values; fP ≤ 0.05 compared with day 30 STZ control values. All the above set of experiments was done in triplicate on two separate occasions (n = 6), and values are expressed as mean ± SEM. (C and D) Body weight of Wistar rats of different groups. The measurements of body weight were done once in 7 d until the end of the study. All the values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ control. AA, arachidonic acid; COX, cyclooxygenase; LOX, lipoxygenase; STZ, streptozotocin; T1DM, type 1 diabetes mellitus.
Nutrition  , 61-80DOI: ( /j.nut )
Copyright © 2016 Elsevier Inc. Terms and Conditions

9. Fig. 8
Effect of AA and COX and LOX inhibitors on plasma insulin, LXA4 and TNF-α levels in STZ-induced T1DM animals. (A) Plasma LXA4 levels in different groups of animals estimated at the end of the study (day 30). (B) Plasma insulin levels in different groups of animals estimated on day 30. All values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ control (T1DM group). (C) Plasma TNF-α levels in different groups of animals estimated once in 10 d until the end of the study (day 30). All values are expressed as mean ± SEM. aP ≤ 0.05 compared with the day 10 control; bP ≤ 0.05 compared with the day 20 control; cP ≤ 0.05 compared with the day 30 control; dP ≤ 0.05 compared with the day 10 STZ control; eP ≤ 0.05 compared with the day 20 STZ control; fP ≤ 0.05 compared with the day 30 STZ control. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ. All the above studies were done wherein each group consisted of n = 6 and all values are expressed as mean ± SEM. AA, arachidonic acid; COX, cyclooxygenase; LOX, lipoxygenase; LX4A, lipoxin A4; STZ, streptozotocin; T1DM, type 1 diabetes mellitus; TNF, tumor necrosis factor.
Nutrition  , 61-80DOI: ( /j.nut )
Copyright © 2016 Elsevier Inc. Terms and Conditions

10. Fig. 9
Effect of LXA4 on STZ-induced T1DM. (A) Protocol of the study. After 7 d of acclimatization, animals received 60 ng/mL LXA4 intraperitoneally for 5 d and 45 mg/kg body weight of STZ only on day 1. (B) Plasma glucose levels. Plasma glucose estimation was performed once in 10 d until the end of the study. All values are expressed as mean ± SEM. aP ≤ 0.05 compared with day 10 control values; bP ≤ 0.05 compared with day 20 control values; cP ≤ 0.05 compared with day 30 control values; dP ≤ 0.05 compared with day 10 STZ values; eP ≤ 0.05 compared with day 20 STZ values; fP ≤ 0.05 compared with day 30 STZ values. *P ≤ 0.05 compared with untreated control; †P ≤ 0.05 compared with STZ control. All the above set of experiments were done in triplicate on two separate occasions (n = 6) and values are expressed as mean ± SEM. (C) Body weight of experimental animals of different groups. Body weights were taken once in 7 d until day 30. All the values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ. (D) Plasma LXA4 levels measured on day 30 of the study. (E) Plasma insulin levels. Plasma insulin levels were estimated on day 30. All values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control; †P ≤ 0.05 compared with STZ. LXA4, lipoxin A4; STZ, streptozotocin; T1DM, type 1 diabetes mellitus.
Nutrition  , 61-80DOI: ( /j.nut )
Copyright © 2016 Elsevier Inc. Terms and Conditions

11. Fig. 10
Plasma TNF-α, gene expression, and protein expression studies in STZ-induced T1DM. (A) Plasma TNF-α levels. Plasma TNF-α measurement was done once every 10 d until the end of the study (day 30). All values are expressed as mean ± SEM. aP ≤ 0.05 compared with day 10 control; bP ≤ 0.05 compared with day 20 control; cP ≤ 0.05 compared with day 30 control; dP ≤ 0.05 compared with day 10 STZ control; eP ≤ 0.05 compared with day 20 STZ control; fP ≤ 0.05 compared with day 30 STZ control. All the above studies were done wherein each group consisted of n = 6 and all values are expressed as mean ± SEM. (B and C) Gene expression studies in the pancreatic tissue. Pancreatic tissue was collected on day 30. The percentage of change in the expression of genes: Pdx1, NF-κB, IKB, and β-actin were studied in various groups by using semi quantitative polymerase chain reaction method. The equality of sample loading was confirmed by β-actin gene expression. (D and E) Protein expression studies in the pancreatic tissue. Total protein extracted from the pancreatic tissue samples collected on day 30 of the study was used for Western blots for Nrf2, Glut2, COX2, iNOS, and β-actin measurements. Equality of loading of the samples was confirmed by β-actin protein expression. All values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ. All the above studies were done wherein each group consisted of n = 6, and values are expressed as mean ± SEM. STZ, streptozotocin; T1DM, type 1 diabetes mellitus; TNF, tumor necrosis factor.
Nutrition  , 61-80DOI: ( /j.nut )
Copyright © 2016 Elsevier Inc. Terms and Conditions

12. Fig. 11
Blood glucose, plasma LXA4 and Insulin levels in STZ induced T2DM animal model. (A) Protocol of the study: After 7 d of acclimatization, T2DM was induced by intraperitoneal administration of STZ 175 mg/kg of body weight, which is considered day 1 of the study. LXA4 60 ng/animal was given intraperitoneally on day 1 (the day STZ was administered) and daily for 5 d. (B) Plasma glucose levels. Plasma glucose was estimated once in 10 d until day 30, the day study was concluded. All values are expressed as mean ± SEM. aP ≤ 0.05 compared with control values of day 10; bP ≤ 0.05 compared with control values of day 20. cP≤ 0.05 compared with control values of day 30; dP ≤ 0.05 compared with STZ values of day 10; eP ≤ 0.05 compared with STZ values of day 20; fP ≤ 0.05 compared with STZ values of day 30. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ control. All the above set of experiments was done in triplicate on two separate occasions (n = 6), and values are expressed as mean ± SEM. (C) Body weight of experimental animals of different groups. Body weight measurements were taken once in 7 d until the end of the study. All the values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control; †P ≤ 0.05 compared with STZ control. (D) Plasma LXA4 levels measured on day 30 of the study. (E) Plasma insulin levels measured on day 30 of the study. All values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control; †P ≤ 0.05 compared with STZ. LXA4, lipoxin A4; STZ, Streptozotocin; T2DM, type 2 diabetes mellitus; TNF, tumor necrosis factor.
Nutrition  , 61-80DOI: ( /j.nut )
Copyright © 2016 Elsevier Inc. Terms and Conditions

13. Fig. 12
Plasma TNF-α concentrations, gene, and protein expression studies. (A) Plasma TNF-α levels. Plasma TNF-α measurement was done once every 10 d until the end of the study. All values are expressed as mean ± SEM. aP ≤ 0.05 compared with the control on day 10; bP ≤ 0.05 compared with the control on day 20; cP ≤ 0.05 compared with the control on day 30; dP ≤ 0.05 compared with the STZ control on day 10; eP ≤ 0.05 compared with the STZ control on day 20; fP ≤ 0.05 compared with the STZ control on day 30. All the above studies were done wherein each group consisted of n = 6, and all values are expressed as mean ± SEM. (B and C) Gene expression studies in the pancreatic tissue. The pancreatic tissue was collected at the end of the experiment. The percentage of change in the expression of genes: Pdx1, NF-κB, IKB, and β-actin were studied using semi quantitative polymerase chain reaction method. The equality of sample loading was confirmed by β-actin gene expression. (D and E) Gene expression studies in adipose tissue. The adipose tissue was collected at the end of the experiment. The percentage of change in the expression of genes: LPCLN2 (lipocalin 2), NF-κB, IKB, and β-actin were studied in various groups of animals using semiquantitative polymerase chain reaction method. The equality of sample loading was confirmed by β-actin gene expression. All values are expressed as mean ± SEM. *P ≤ 0.05 compared with untreated control, †P ≤ 0.05 compared with STZ control. *P ≤ 0.05 compared with untreated control. †P ≤ 0.05 compared with STZ control. All the above studies were done wherein each group consisted of n = 6, and values are expressed as mean ± SEM. (F) Calculated ratio between the activities of NF-κB and IKK in the pancreas and adipose tissue of STZ-induced T1- and T2DM that were treated with LXA4. LXA4, lipoxin A4; STZ, streptozotocin; T2DM, type 2 diabetes mellitus; TNF, tumor necrosis factor.
Nutrition  , 61-80DOI: ( /j.nut )
Copyright © 2016 Elsevier Inc. Terms and Conditions