JOURNAL OF DIABETES, ENDOCRINOLOGY AND METABOLIC DISEASES

vol.

Vol. 44 No. 3 (pp. 67 - 96) 2015 / Zagreb, August 2016

44 no.

3

p.p.

67-96 2015

Journal of Diabetes, Endocrinology and Metabolic Diseases VUK VRHOVAC UNIVERSITY CLINIC, ZAGREB, DAMA - DIABETOLOGY ALUMNI MEDICAL ASSOCIATION

CONTENTS ORIGINAL RESEARCH ARTICLES HEAT PRECONDITIONING AND ASPIRIN TREATMENT MODULATE DIABETIC RAT KIDNEY METABOLISM Nataša Cipanovska, Mirsada Dervisevik, Maja Dimitrovska, Suzana DinevskaKjovkarovska, Biljana Miova......................................................................................... 67 BENEFICIAL EFFECTS OF VISCOUS FIBER BLEND, AMERICAN AND RED KOREAN GINSENGS, SALVIA HISPANICA L. (SALBA, CHIA), OILY GRAIN, ON CARDIOVASCULAR RISK FACTOR MANAGEMENT IN TYPE 2 DIABETIC PATIENTS Saša Magaš, Lea Smircˇic´ Duvnjak, Jelena Miocˇic´....................................................... 81 REVIEW

UDC 616.379-008.67.43

ISSN 0351-0042

MANAGEMENT OF THYROID NODULES Mario Škugor ................................................................................................................ 87

MEDICAL SCIENTIFIC JOURNAL OF THE VUK VRHOVAC INSTITUTE UNIVERSITY CLINIC FOR DIABETES, ENDOCRINOLOGY AND METABOLIC DISEASES SCHOOL OF MEDICINE, UNIVERSITY OF ZAGREB CROATIAN MEDICAL ASSOCIATION, CROATIAN SOCIETY FOR ENDOCRINOLOGY AND DIABETOLOGY DIABETOLOGY ALUMNI MEDICAL ASSOCIATION VUK VRHOVAC UNIVERSITY CLINIC, ZAGREB

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VOLUME 44, NUMBER 3, 1, 2015

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Original Research Article

Department of Experimental Physiology and Biochemistry, Institute of Biology, Faculty of Natural Sciences and Mathematics, St Cyril and Methodius University, Skopje, R. Macedonia

HEAT PRECONDITIONING AND ASPIRIN TREATMENT MODULATE DIABETIC RAT KIDNEY METABOLISM Nataša Cipanovska, Mirsada Dervisevik, Maja Dimitrovska, Suzana Dinevska-Kjovkarovska, Biljana Miova

Key words: diabetes mellitus, heat preconditioning, aspirin, carbohydrate metabolism, antioxidative defense, kidney, rats

SUMMARY Considering that heat preconditioning (HP) is a powerful adaptive and protective phenomenon, we hypothesize that carbohydrate- and oxidativerelated changes in streptozotocin (STZ)-diabetic rats are less pronounced in heat exposed animals. Moreover, the common effect of HP with aspirin (ASA) pretreatment was investigated, since ASA modulates the thermotolerant state of the cells, lowers glucose concentration and attenuates oxidative stress in diabetic state. In this sense, HP protocol (45 min at 41±0.5 ºC, followed by 24 h recovery at room temperature) was combined with ASA pretreatment (100 mg/kg b.w.) in STZ (55 mg/kg)-diabetic rats. HPCorresponding author: Prof. Biljana Miova, PhD, Department of Experimental Physiology and Biochemistry, Institute of Biology, Faculty of Natural Sciences and Mathematics, St Cyril and Methodius University, Arhimedova 3, 1000 Skopje, R. Macedonia E-mail: [email protected]; [email protected]

Diabetologia Croatica 44-3, 2015

diabetic animals manifested lower serum and kidney glucose (Glu) and glucose-6-phosphatase (G6Pase) activity, higher glycogen (Glk) content, as well as increased antioxidative potential, i.e. increased concentrations of glutathione (GSH), increased glutathione reductase (GR) and catalase (Cat) and decreased glutathione peroxidase (GPx) activity as compared with normothermic diabetic animals. ASApretreatment of HP-diabetic animals led to timedependent modifications of carbohydrate metabolism. Also, there was reduction of GSH, GR and Cat, and elevation of GPx in ASA-pretreated HP-diabetic animals. In conclusion, HP and aspirin alleviate diabetic complications and increase antioxidative potential in rats, manifesting protective effects against diabetic disturbances.

INTRODUCTION Diabetes mellitus (DM) is a metabolic disorder of multifactorial etiology characterized by chronic hyperglycemia with disturbance of the whole body metabolism. It is well known that the primary stressors that elicit this response in DM are hyperglycemia and oxidative stress (1). A very pronounced phenomenon in DM is also altered expression of molecular

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Nataša Cipanovska, Mirsada Dervisevik, Maja Dimitrovska, Suzana Dinevska-Kjovkarovska, Biljana Miova / HEAT PRECONDITIONING AND ASPIRIN TREATMENT MODULATE DIABETIC RAT KIDNEY METABOLISM

chaperones since long-lasting hyperglycemia causes significant modifications of cellular proteins (2). Low levels of heat shock proteins (HSPs) contribute to an impaired stress response, protein glycation and oxidation, free radical formation, protein aggregation and inflammation, and may be a clue to the etiology of the disease itself (3). The most favorable condition to increase the production of HSP is acute heat stress (HS). In this sense, different organisms/cells previously exposed to sublethal HS raise greater resistance to the effects of higher intensity stress such as hypoxia, ischemia/ reperfusion (4), or strong cytotoxic agent such as the diabetogenic agent streptozotocin (STZ) (5,6), and develops a protective mechanism, known as heat preconditioning (HP). An abundant body of evidence points to the beneficial effect of heat preconditioning for DM (7,8). Considering the above, one of the aims of this investigation was to assess the effects of HP on STZ-induced diabetic disturbances in rats. Besides the physiological induction of HSP by HP, there are few pharmacological inductors of HSP. Among them, nonsteroidal anti-inflammatory drugs (NSAIDs) such as sodium salicylate, aspirin and indomethacin have been reported to modulate the heat shock response (HSR) in different organisms (9,10). Aspirin is known to diminish endogenous oxidant stress and enhance resistance to exogenous peroxide, both likely to be mediated by activation of antioxidant defense (11) and by blockage of oxidative status in diabetic rats (12). Also, it is known to reduce blood glucose level in diabetic rats (13). Taking in consideration the above, additionally, we investigated the possible aspirin-induced modifications in normothermic and HP-diabetic animals. The investigations were carried out on rat kidney since in diabetes, significant damage also occurs in tissues where the entry of glucose is not regulated by insulin, such as the kidney (14). Diabetes causes increase of renal glucose uptake, which in turn results in accumulation of glycogen found in diabetic kidney

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(15) and increases oxidative stress in rat kidney (16). Also, there is evidence that in rat kidney sublethal HS results in rapid cytosolic accumulation of the cytoprotectant HSP720, and may be important in mediating cell repair or increasing resistance to subsequent injury (17). Thus, the effects of HP alone and in combination with ASA were estimated through changes in carbohydrate-related and antioxidative defense enzymes and substrates in rat kidney.

MATERIALS AND METHODS Animals and tissue procedures The study was performed at the Department of Experimental Physiology and Biochemistry, Institute of Biology, Faculty of Natural Sciences and Mathematics, St Cyrilus and Methodius University, Skopje, R. Macedonia. The study was carried out in male Wistar laboratory rats (3-4 months old) from our in-house breeding facility (Institute of Physiology and Biochemistry of Natural Sciences and Mathematics, Skopje), in a controlled environment at 12-hour light regimen (06 -18 p.m. light) and feeding ad libitum throughout the study. All experimental animals were anesthetized with Nathiopental narcosis (45 mg/kg) and sacrificed using a standard laparothomic procedure. The sacrifice was always performed at the same time of day (9.0010.00 a.m.) and feed was removed from cages 6 hours earlier. Immediately upon abdominal cavity opening, the isolated kidney was washed with cold saline solution and immersed in liquid nitrogen. Tissues were kept at -80 °C until analyses. Tissue homogenate of whole kidney was prepared in a suitable medium for homogenization and further used to perform appropriate analyses. Homogenization was performed with ultrasonic homogenizer (Cole-Parmer Instrument 4710), in several cycles of 5 seconds, always in ice (at a temperature of 0-4 °C). All procedures followed the Guidelines of the European Community Committee on Care and Use of Laboratory Animals and Good Laboratory Practice.


Study design and treatments The animals were divided into two general groups: control and diabetic. Control group was divided into three subgroups: control animals (C), heatpreconditioned control animals (HC) and heatpreconditioned control animals pretreated with ASA (AHC). Diabetic group was divided into six subgroups: control diabetic groups (D2 and D14, sacrificed 2 or 14 days after STZ administration, respectively); heatpreconditioned diabetic groups (HD2 and HD14, sacrificed 2 or 14 days after STZ administration, respectively), and heat-preconditioned diabetic groups pretreated with ASA (AHD2 and AHD14, sacrificed 2 or 14 days after STZ administration, respectively).

Heat preconditioning was done by exposure of animals in special temperature-controlled chambers (45 min at 41±0.5 ºC), followed by 24-h recovery at room temperature (20±2 ºC). The duration of diabetes in our experiment was defined by two criteria: minimum period for manifestation of the effect of STZ administered (48 h) and optimal period for the development of diabetic complications (14 days). The induction of experimental diabetes was performed by a single intraperitoneal injection of STZ (55 mg/kg body weight), freshly dissolved in 0.1M citrate buffer, pH 4.5. All animals with clear diabetic symptoms (fasting glycemia levels higher

Table 1. Study parameters according to experimental groups of animals

ASA = acetylsalicylic acid; STZ = streptozotocin; C = control animals; HC = heat preconditioned control animals; AHC = heat-preconditioned control animals pretreated with ASA; D2 and D14 = diabetic animals (sacrificed 2 or 14 days after STZ administration, respectively); HD2 and HD14 = heat-preconditioned diabetic animals (sacrificed 2 or 14 days after STZ administration, respectively); AHD2 and AHD14 = heat-preconditioned diabetic animals pretreated with ASA (sacrificed 2 or 14 days after STZ administration, respectively).


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than 15 mmol/L) 24-48 hours after the induction of experimental diabetes were used for the purpose of this experiment. Aspirin (acetylsalicylic acid (ASA), Sigma-Aldrich) was freshly dissolved in water (as 100 mg/kg b.w. solution). Subsequently, sodium carbonate crystals were slowly added, until the ASA crystals had dissolved (the pH of the solution remained just below 7.0 (18)) and administered intraperitoneally in a 0.5mL volume (10,19), one hour before exposure to heat stress.

Biochemical tests Carbohydrate-related enzymes and substrates Serum glucose (Glc) concentration was determined using a commercially available enzymatic colorimetric test (GOD-PAP method, Human, Germany). Kidney glycogen (Glk), glucose (Glu) and glucose-6phosphate (G6P) concentrations were determined in perchlorate homogenates and neutralized with 5M K2CO3. We measured the production of NADPH at 340 nm in a reaction catalyzed by glucose-6-phoshate dehydrogenase (20). The specific activity of glucose6-phosphatase (G6Pase) was estimated according to the method of Hers et al. (21). For interpretation of the activity as a specific enzyme activity (nmol Pi./min/ mg proteins) for GFa, HK and PFK (U/mg protein), the total quantity of the proteins was measured by the method of Lowry et al. (22). Antioxidative defense enzymes and substrates Kidney catalase activity was assessed following Aebi’s method (23), which is based on spectrophotometric measurement of absorbance decrease due to consumption of H2O2. The principle of reaction for determination of glutathione reductase (GR) is based on measuring the reduction in absorbance that occurs as a result of NADPH oxidation (adaptation to SigmaAldrich determination kit). Estimation of glutathione peroxidase (GPx) activity in the kidney was performed by measuring the consumption of NADPH as a result of its oxidation to NADP+ (adaptation to

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Sigma-Aldrich determination kit). The method for determining glutathione (GSH) concentration is based on the reduction of 5,5’-ditiobis (2-nitrobensoic acid) (DTNB) in the 5-thio-nitrobensoic acid (TNB) (Sigma Aldrich determination kit).

Statistics Results are presented as means ± SD. To assess statistical differences among the groups, we used oneway ANOVA with Neuman-Keuls post-hoc test. Only significant coefficients of correlation are presented in figures. In all tests, a probability level of p<0.05 was used as a significant difference. The overall statistical data processing was performed using the Statgraph for Windows 3.0 statistical program.

RESULTS Rectal temperature (effects of HP, ASA and ASA+HP) Our measurements showed a significant increase of rectal temperature in HP-animals by about 3.7 °C, which returned to baseline values after 24 h recovery at room temperature. Aspirin treatment alone rose rectal temperature by about 1 °C one hour after treatment. Aspirin pretreatment in combination with HP (ASA+HP) caused additional increase of rectal temperature (total increment by about 4.4 °C). Finally, when animals recovered for 24 h at room temperature, normalization of rectal temperature was observed (results not shown). Carbohydrate-related enzymes and substrates Changes in carbohydrate metabolism are illustrated in Figure 1: (A) kidney glucose (Glu) concentration; (B) serum glucose (Glu) concentration; (C) glycogen (Glk) content; (D) glucose-6-phosphate (G6P) concentration; and (E) glucose-6-phosphatese (G6Pase) activity. It is evident that HP of control animals (C:HC) caused significant increase only in G6Pase activity and no significant changes in other estimated parameters.


Our results showed that STZ administration (C:D2, C:D14) resulted in a statistically significant (2-fold) increase in serum and kidney Glu concentration (9to 10-fold increase), regardless of the duration of diabetes. Also, the results indicated that STZ-induced DM caused an increase in kidney Glk content and G6P concentration, as well as in G6Pase activity.

of Glk content regardless of the duration of diabetes. Pretreatment of HP-intact animals with ASA (HC:AHC) resulted in a significant increase in kidney Glu concentration and G6Pase activity, and a decreased G6P concentration. Pretreatment of HP-diabetic animals with ASA (HD2:AHD2, HD14: AHD14) led to a decrease in serum Glu and increase in kidney Glu concentration. As for the Glk content, ASA+HP resulted in a reduction in 2d-diabetic animals, but an increase in 14d-diabetic animals. These treatments caused a decrease in the concentration of G6P and nonsignificant changes in G6Pase activity.

Heat preconditioning of diabetic animals (D2:HD2, D14:HD14) resulted in increased G6Pase activity, followed by lower serum Glu and especially lower kidney Glu concentration as compared with diabetic controls. By contrast, HP caused more evident increase

Figure 1. Changes in carbohydrate-related enzymes and substrates in kidney of heat preconditioned control and diabetic rats treated with aspirin: (A) kidney glucose (Glu) concentration; (B) serum glucose (Glu) concentration; (C) glycogen (Glk) content; (D) glucose-6-phosphate (G6P) concentration; and (E) glucose-6phosphatese (G6P-ase) activity.

Serum glucose concentration 25

mmol Glu/L

20

a

b

a

d

15 10 5 0

B


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mg Glk/g tissue

Glycogen content 10 9 8 7 6 5 4 3 2 1 0

d

b

b

a

d a

C Glucose-6-phosphate concentration µmol G6P/ g tissue

0,04

a

a

0,03

d 0,02

c

0,01 0,00

D

Glucose-6-phosphatase activity 160

c

140

U/ mg prot

120 100 80

b

a a

a

b

60 40 20 0

E

ASA = acetylsalicylic acid; STZ = streptozotocin; C = control animals; HC = heat preconditioned control animals; AHC = heat-preconditioned control animals pretreated with ASA; D2 and D14 = diabetic animals (sacrificed 2 or 14 days after STZ administration, respectively); HD2 and HD14 = heat-preconditioned diabetic animals (sacrificed 2 or 14 days after STZ administration, respectively); AHD2 and AHD14 = heat-preconditioned diabetic animals pretreated with ASA (sacrificed 2 or 14 days after STZ administration, respectively); significant difference (p <0.050): (a) relative to control animals (C:HC, C:D2, C:D14); (b) relative to diabetic animals (D2:HD2, D14:HD14, respectively); (c) relative to heat preconditioned intact animals (HC:AHC); (d) relative to heat-preconditioned diabetic animals (HD2:AHD2, HD14:AHD14, respectively).

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Antioxidative defense enzymes and substrates Changes in antioxidative defense enzymes and substrates are presented in Figure 2: (A) glutathione (GSH) concentration; (B) glutathione reductase (GR) activity; (C) glutathione peroxidase (GPx) activity; and (D) catalase (Cat) activity.

only significant in 14d-diabetic animals (where an 83% reduction was recorded), followed by a decrease of GR and CAT activity. By contrast, there was a progressive increase of GPx activity in 14d-diabetic animals. Heat preconditioning of diabetic animals (D2:HD2, D14:HD14) led to a significant increase in the concentration of GSH, as well as in GR and CAT activity, but a decrease of GPx activity.

Our results showed that HP of control animals (C:HC) resulted in a significant increase in Cat activity and GSH concentration, but as for the other parameters, HP did not cause significant changes.

Pretreatment of HP-intact animals with ASA (HC:AHC) caused significant reduction of GSH concentration and CAT activity, but an increase in GPx activity.

The results showed that STZ (C:D2, C:D14) caused a decrease in GSH concentration, but the changes were

Figure 2. Changes in antioxidative-defense enzymes and substrates in kidney of heat-preconditioned control and diabetic rats treated with aspirin: (A) glutathione (GSH) concentration; (B) glutathione reductase (GR) activity; (C) glutathione peroxidase (GPx) activity; and (D) catalase (Cat) activity. Gluthatione concentration

nm GSH/mg tissue

7 5 4

b

b

6

d

a c

3 2

a

1 0

A

Gluthatione reductase activity 250

U/mg prot

200 150

b

b a

d a

100 50 0

B


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Gluthatione peroxidase activity 140

U/mg prot

120

c

b

100

d

a

b

80 60 40 20 0

C

Catalase activity 16 14

a b

U/mg prot

12 10 8 6

b

d

c a

a

4 2 0

D

ASA = acetylsalicylic acid; STZ = streptozotocin; C = control animals; HC = heat preconditioned control animals; AHC = heat-preconditioned control animals pretreated with ASA; D2 and D14 = diabetic animals (sacrificed 2 or 14 days after STZ administration, respectively); HD2 and HD14 = heat-preconditioned diabetic animals (sacrificed 2 or 14 days after STZ administration, respectively); AHD2 and AHD14 = heat-preconditioned diabetic animals pretreated with ASA (sacrificed 2 or 14 days after STZ administration, respectively); significant difference (p <0.050): (a) relative to control animals (C:HC, C:D2, C:D14); (b) relative to diabetic animals (D2:HD2, D14:HD14, respectively); (c) relative to heat preconditioned intact animals (HC:AHC); (d) relative to heat-preconditioned diabetic animals (HD2:AHD2, HD14:AHD14, respectively). Pretreatment of HP-diabetic animals with ASA resulted in a decrease of GSH concentration, GR and Cat activity, along with an increase of GPx activity only in 2d-diabetic animals (HD2:AHD2).

DISCUSSION Effect of heat preconditioning on the rat’s kidney carbohydrate metabolism and antioxidative defense

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Evidence from many investigations suggests that carbohydrate metabolism (24,25) and oxidative state (26) are altered during and after HS. Heat stress is known to initiate sympatico-adrenal responses in the body (27). Further on, increased catecholamines cause elevation of renal gluconeogenesis, first of all through increased availability of the precursors (28) and increase of glucose-6-phospahtase activity (29). The results of our study indicated that heat preconditioning followed by 24-h recovery at room


temperature led only to increased kidney G6Pase activity (Fig. 1E), with no changes at the substrate level. It might be that changes in the estimated carbohydrate-related parameters are not so evident since the animals recovered 24 h after HS. On the other hand, increased catecholamines during HS result in increased release of liver GSH (30) and since the kidney is the only tissue that is capable for intake of extracellular GSH (31), we found increased GSH in our study (Fig. 2A). Increased Cat in our study (Fig. 2D) implicated elevated antioxidative potential of the organism, since this enzyme has an important role in acquiring oxidative tolerance and adaptive mechanisms of the cell (32). Nevertheless, we suppose that the changes in carbohydrate metabolism and oxidative defense after HS are rapid and reversible, so, in our investigation, the period of recovery of 24 h was sufficient for normalization of the levels of the parameters analyzed. Effect of streptozotocin on the rat kidney carbohydrate metabolism and antioxidative defense Renal gluconeogenesis is increased (33,34) and stimulation of the gluconeogenic enzyme glucose-6phosphatase (Fig. 1E) is evident in the condition of diabetes (35,36). Besides the increased concentration of serum and kidney Glu (Fig. 1A and 1B), renal glucose uptake is also evident in diabetes (34,37), leading to a significant deposition of Glk, as observed in our study (Fig. 1C). Kidney Glk levels were approximately 30fold higher in diabetic animals due to the increased amounts of total glycogen synthase and its activator G6P (38), and prolonged hyperglycemia is the sole driving force for this phenomenon (39). The possible link between oxidative stress and impaired glucose metabolism in diabetic condition is provided by the data that increased G6Pase (Fig. 1E) and decreased G6PDH activity cause decreased influx of G6P into the pentose-phosphate cycle, which results


in a decreased production of NADPH (40). NADPH is a critical cofactor for maintenance of the active state of Cat (41,42), serves as a cofactor for GR reduction of GSSG to its reduced form, and is a substrate for GPx (43). Considering that GSH metabolism and distribution of glutathione S-transferase in tissues of diabetic rats are crucial for the etiology, pathology and prevention of diabetes (44), greater reduction of GSH levels that we found in our study (about 83% in 14d after STZ administration, Fig. 2A) is a clear indication of impaired oxidative status in diabetic condition. The increased GPx activity and decreased Cat activity (Fig. 2C and 2D) suggest that there could be a compensatory mechanism between the antioxidant enzymes in response to oxidative stress (45). Effect of heat preconditioning on the diabetic rat kidney carbohydrate metabolism and antioxidative defense The most evident change caused by HP of diabetic animals was increased accumulation of Glk (Fig. 1C) as a result of rapid mobilization of tissue and serum Glu (Fig. 1A and 1B) through increased G6Pase activity (Fig. 1E). Our assumption is that the protective mechanism might be based on the induction of heat shock proteins (HSP) by previous HS, which act not only as molecular chaperones, but also as housekeeping molecules (46). Najemnikova et al. (6) report that 24 hours following HS there is an increased HSP72 content in the heart, kidney and liver from diabetic animals compared with the same tissues from heat-stressed nondiabetic animals. According to Amici et al. (47), there is a positive correlation between the level of HSP72 and circulating levels of insulin and Glu, and its uptake and oxidation in peripheral tissues. The fact that the induction of HSP70 in intact animals is fully expressed for 24 h-48 h after stress exposure (47) could be a possible explanation for the observed changes 2d after STZ. Moreover, the most common mechanism of the body to fight oxidative stress is the induction of protective

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genes, including those belonging to the HSP family (48,49). In our results, heat preconditioning prevented diabetes-induced reduction of Cat, GR and GSH (Fig. 2A, 2B and 2D). Due to the fact that oxidative damage and protein glycation are the major causes of diabetic complications, the elevation of chaperone capacity (i.e. HSP72) in diabetic patients seems to be an important strategy for protection of proteins against these harmful changes and conservation of their native molecule structures (50). Effect of aspirin treatment and heat preconditioning on control and diabetic rat kidney carbohydrate metabolism and antioxidative defense Concerning the effects of ASA-pretreatment of heat preconditioned control animals (HS:AHS), we observed only significant increase of G6Pase activity and decrease of G6P concentration, and nonsignificant changes in glycogen and glucose concentration. More evident effects were observed in antioxidative defense enzymes and substrates, i.e. decrease of GSH and Cat and increased GPx activity (Fig. 2A, 2C, 2D), but all of them reaching almost control values. Data from other researches indicate that ASA treatment results in a decrease of fasting serum Glu level in healthy patients (51) and also attenuates oxidative stress (52). In addition, we found significant increase of rectal temperature 1 h after aspirin treatment (result not shown), which could be the main trigger for increased HSP production. Namely, ASA in combination with heat stress is a co-inducer of HSP both in erythroleukemic and HeLa cells (47) and in rats (10). Our assumption is that the modulation of HS response by ASA, associated with the ability of this drug to influence the establishment of a thermotolerant state in cells that have undergone hyperthermia (47), is the main reason for establishing control values of all study parameters. Estimating ASA pretreatment of HP-diabetic animals, it is evident that ASA treatment caused a less pronounced hyperglycemic condition, i.e. lower serum Glu concentration (Fig. 1B). Still, we observed timedependent changes: 2d after STZ administration there

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was evident increase in kidney Glu, followed by lower accumulation of kidney Glk content. By contrast, 14d after STZ administration, there was an intensified intake of serum Glu, but kidney glucose accumulated in glycogen stores. The positive effects of ASA on hyperglycemic state in terms of diabetes have been previously described (53,54), but these data still do not provide complete view of the effect of ASA on diabetic kidney. Concerning heat preconditioning, as we mentioned before, thermotolerance in intact animals was fully expressed for 24 h-48 h of stress exposure and decreased later on (47). It might be that in the period of 24 h-48 h after HS, the cells are more protected from the diabetogenic effect of STZ, resulting in different blood control between different diabetic groups. As for the parameters of oxidative status, our results showed that pretreatment with ASA caused partial restoration of the activity of all three antioxidant enzymes in diabetic animals almost to control values (Fig. 2B, 2C, 2D) and lower concentration of GSH in diabetic hyperthermic animals, regardless of the duration of diabetes (Fig. 2A). Namely, ASA is known to have antioxidant capacity by inhibiting the formation of advanced glycation end products (AGEs) and thereby reduces oxidative stress in diabetic nephropathy (55). There is an implication for protected renal ischemia/reperfusion injury by heat preconditioning (56) and improved kidney graft function and survival in rats (57), both of them by induction of HSPs. In this sense, we suppose that this antioxidative capacity is elicited through the increased production of HSP in heat-exposed organisms. Finally, it can be concluded that heat preconditioning modulates the effect of experimental diabetes by alleviating diabetic complications and increasing antioxidative potential in experimental animals. On the other hand, treatment with aspirin minimizes toxic effect of STZ. Further investigation of the induction and expression of HSP is needed for clarifying the effects of heat preconditioning on modifying metabolic changes caused by diabetes.


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10. Fawcett JW, Xu Q, Holbrook KJ. Potentiation of heat stress-induced HSP70 expression in vivo by aspirin. Cell Stress Chaperones. 1997;2(2): 104-109. 11. Ayyadevara S, Bharill P, Dandapat A, Hu C, Khaidakov M, Mitra S, et al. Aspirin inhibits oxidant stress, reduces age-associated functional declines, and extends lifespan of Caenorhabditis elegans. Antioxid Redox Signal. 2013;18: 481-490. 12. Caballero F, Gerez E, Batlle A, Vazquez E. Preventive aspirin treatment of streptozotocin induced diabetes: blockage of oxidative status and reversion of heme enzymes inhibition. Chem Biol Interact. 2000;126:215-225. 13. Jafarnejad A, Bathaie SZ, Nakhjavani M, Hassan MZ. Investigation of the mechanisms involved in the high-dose and long-term acetylsalicylic acid therapy of type I diabetic rats. J Pharmacol Exp Ther. 2008;324:850-857. 14. Pallavi VL, Nadnula R, Sivakami S. Oxidative stress and gene expression of antioxidant enzymes in the renal cortex of streptozotocin-induced diabetic rats. Mol Cell Biochem. 2003;243: 147-152. 15. Meyer C, Stumvoll M, Nadkarni V, Dostou J, Mitrakou A, Gerich J. Abnormal renal and hepatic glucose metabolism in type 2 diabetes mellitus. J Clin Invest. 1998;102:619-624.

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25. Miova B, Dinevska-Kjovkarovska S, Dzimrevska A, Mitev S. Prior heat stress induces moderation of diabetic alterations in glycogen metabolism of rats. Cent Eur J Biol. 2014;9(3):249-259.

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26. Heise K, Puntarulo S, Nikinamaa M, Abele DO, Portner H. Oxidative stress during stressful heat exposure and recovery in the North Sea eelpout Zoarces viviparous L. J Exp Biol. 2006;209:353 363.

18. Kelton JG, Hirsh J, Carter CJ, Buchanan MR. Thrombogenic effect of high-dose aspirin in rabbits: relationship to inhibition of vessel wall synthesis of prostaglandin 12-like activity. J Clin Invest. 1978;62:892-895. 19. Locke M, Atance J. The myocardial heat shock response following sodium salicylate treatment. Cell Stress Chaperones. 2000;5(4):359-368. 20. Keppler D, Decker K. Glycogen determination with amyloglucosidase. In: Bergmeyer HU (Ed). Methods of Enzymatic Analysis, Vol 3. New York: Academic Press, 1974; p 1127-1131. 21. Hers HG, Levine R, Luft R. Glycogen storage disease. In Levine R, Luft R. (Eds). Advances in Metabolic Disorders. New York- London: Academic Press, 1964; p 36-85. 22. Lowry OH, Rosebrough JN, Farr LA, Randall JR. Protein measurement with the folinphenol reagent. J Biol Chem. 1951;193:265-275. 23. Aebi H. Catalase. In: Bergmeyer HU (Ed). Methods of Enzymatic Analysis. New York: Academic Press, 1974; pp 673-677. 24. Streffer C. Aspects of metabolic change after hyperthermia. Recent Results Cancer Res. 1998;107:7-16.

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27. Edens FW, Siegel HS. Adrenal response in high and low ACTH response lines of chickens during acute heat stress. Gen Comp Endocrinol. 1975;25:64. 28. Chu CA, Singer DA, Neal DW, Cherrington AD. Direct effects of catecholamines on hepatic glucose production in conscious dog are due to glycogenolysis. Am J Physiol. 1996 Jul; 271 (1 Pt 1):E127-137. 29. Bady I, Zitoun C, Guignot L, Mithieux G. Activation of liver glucose-6-phosphatase in response to insulin-induced hypoglycemia or epinephrine infusion in the rat. J Clin Investig. 2002;282(4):905-910. 30. Sies H, Graf P. Hepatic thiol and glutathione efflux under the influence of vasopressin, phenylephrine and adrenaline. Biochem J. 1985;226:545-549. 31. Wu G, Fang YZ, Yang S, Lupton JR, Turner ND. Glutathione metabolism and its implications for health. J Nutr. 2004;134:489-492. 32. Mocanu MM, Steare SE, Evans MCW, Nugent JH, Tellon DM. Heat stress attenuates free radical release in the isolated perfused rat heart. Free Rad Biol Med. 1993;15:459-463.


33. Garcia-Salguero L, Lupififiez JA. Metabolic adaptation of the renal carbohydrate metabolism. I. Effects of starvation on the gluconeogenic and glycolytic fluxes in the proximal and distal renal tubules. Mol Cell Bioehem. 1998;83:167-178. 34. Meyer C, Stumvoll M, Nadkarni V, Dostou J, Mitrakou A, Gerich J. Abnormal renal and hepatic glucose metabolism in type 2 diabetes mellitus. J Clin Invest. 1998;102:619-624. 35. Eid A, Bodin S, Ferrier B, Delage H, Boghossian M, Martin M, et al. Intrinsic gluconeogenesis is enhanced in renal proximal tubules of Zucker diabetic fatty rats. J Am Soc Nephrol. 2006;17: 398-405. 36. Pari L, Satheesh AM. Effect of pterostilbene on hepatic key enzymes of glucose metabolism in streptozotocin and nicotinamide induced diabetic rats. Life Sci. 2006;79:641-645. 37. Meyer C, Woerle HJ, Dostou JM, Welle SL, Gerich JE. Abnormal renal, hepatic, and muscle glucose metabolism following glucose ingestion in type 2 diabetes. Am J Physiol Endocrinol Metab. 2004;287:E1049-E1056. 38. Khandelwal RL, Zinman SM, Knull HR. The effect of streptozotocin-induced diabetes on glycogen metabolism in rat kidney and its relationship to the liver system. Arch Biochem Biophys. 1979;197:310-316. 39. Nannipieri M, Lanfranchi A, Santerini D, Catalano C, Van de Werve G, Ferrannini E. Influence of long-term diabetes on renal glycogen metabolism in the rat. Nephron. 2001;87(1):50-57. 40. Okwudiri OO, Sylvanus AC, Peace IA. Monosodium glutamate induces oxidative stress and affects glucose metabolism in the kidney of rats. Int J Biochem Res Rev. 2012;2(1):1-11.


41. Gaetani GF, Kirkman HN, Mangerini R, Ferraris AM. Importance of catalase in the disposal of hydrogen peroxide within human erythrocytes. Blood. 1994;84:325-330. 42. Kirkman HN, Rolfo M, Ferraris AM, Gaetani GF. Mechanisms of protection of catalase by NADPH. Kinetics and stoichiometry. J Biol Chem. 1999;274:13908-13914. 43. Xu Y, Osborne BW, Stanton RC. Diabetes causes inhibition of glucose-6-phosphate dehydrogenase via activation of PKA, which contributes to oxidative stress in rat kidney cortex. Am J Physiol Renal Physiol. 2005;289:F1040-F1047. 44. Raza H, Ahmed I, John A. Tissue specific expression and immunohistochemical localization of glutathione S-transferase in streptozotocin induced diabetic rats: modulation by Momordica charantia (karela) extract. Life Sci. 2004;74: 1503-1511. 45. Pallavi VL, Nadnula R, Sivakami S. Oxidative stress and gene expression of antioxidant enzymes in the renal cortex of streptozotocin-induced diabetic rats. Mol Cell Biochem. 2003;243: 147-152. 46. Zhu Q, Xu YM, Wang LF, Zhang Y, Wang F, Zhao J, et al. Heat shock protein 70 silencing enhances apoptosis inducing factor-mediated cell death in hepatocellular carcinoma HepG2 cells. Cancer Biol Ther. 2009;8(9):792-798. 47. Amici C, Rossi A, Santoro GM. Aspirin enhances thermotolerance in human erythroleukemic cells: an effect associated with the modulation of the heat shock response. Cancer Res. 1995;55: 4452-4457.

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48. Kurucz I, Morva A, Vaag A, Eriksson KF, Huang X, Groop L, et al. Decreased expression of heat shock protein 72 in skeletal muscle of patients with type 2 diabetes correlates with insulin resistance. Diabetes. 2002;51:1102-1109. 49. Horowitz M, Eli-Berchoer L, Wapinski I, Friedman N, Kodesh E. Stress-related genomic responses during the course of heat acclimation and its association with ischemic-reperfusion cross-tolerance. J Appl Physiol. 2004;97: 1496-1507. 50. Calabrese V, Scapagnini G, Ravagna A, Colombrita C, Spadaro F, Butterfield DA, et al. Increased expression of heat shock proteins in rat brain during aging: relationship with mitochondrial function and glutathione redox state. Mech Ageing Dev. 2004;125:325-335. 51. Micossi P, Pontiroli AE, Baron SH, Tamayo RC, Lengel F, Bevilacqua M, et al. Aspirin stimulates insulin and glucagon secretion and increases glucose tolerance in normal and diabetic subjects. Diabetes. 1978;27(12):1196-1204. 52. Lapshina EA, Sudnikovich EJ, Maksimchik JZ, Zabrodskaya SV, Zavodnik LB, Kubyshin VL, et al. Antioxidative enzyme and glutathione S-transferase activities in diabetic rats exposed to long-term ASA treatment. Life Sci. 2006;79:1804-1811. 53. Hundal RS, Petersen KF, Mayerson AB, Randhawa PS, Inzucchi S, Shoelson SE, et al. Mechanism by which high dose aspirin improves glucose metabolism in type 2 diabetes. J Clin Invest. 2002;109:1321-1326. 54. Abdin AA, Baalash AA, Hamooda HE. Effects of rosiglitazone and aspirin on experimental model of induced type 2 diabetes in rats: focus on insulin resistance and inflammatory markers. J Diabetes Complications. 2010; 24:168-178.

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55. Edwards JL, Vincent AM, Cheng HT, Feldman EL. Diabetic neuropathy: mechanisms to management. Pharmacol Ther. 2008;120(1):1-34. 56. O’Neill S, Hughes J. Heat-shock protein-70 and regulatory T cell-mediated protection from ischemic injury. Kidney Int. 2013;85:5-7. 57. Redaelli CA, Tien YH, Kubulus D, Mazzucchelli L, Schilling MK, Wagner AC. Hyperthermia preconditioning induces renal heat shock protein expression, improves cold ischemia tolerance, kidney graft function and survival in rats. Nephron. 2002;90(4):489-497.

Original Research Article

Merkur University Hospital, Vuk Vrhovac University Clinic for Diabetes, Endocrinology and Metabolic Diseases

BENEFICIAL EFFECTS OF VISCOUS FIBER BLEND, AMERICAN AND RED KOREAN GINSENGS, SALVIA HISPANICA L. (SALBA, CHIA), OILY GRAIN, ON CARDIOVASCULAR RISK FACTOR MANAGEMENT IN TYPE 2 DIABETIC PATIENTS Saša Magaš, Lea Smirčić Duvnjak, Jelena Miočić Key words: cardiovascular risk factor management, viscous fiber blend, American and Red Korean ginseng, Salvia hispanica L. (salba, chia)

SUMMARY Cardiovascular disease is a major burden in patients with type 2 diabetes mellitus. Therefore, attention is increasingly paid to dietary approaches or interventions that can reduce the risk of cardiovascular disease. Viscous fiber blend, ginseng, oily grain (salba,chia) has been repeatedly shown to attenuate cardiovascular disease risk factors in acute and long term trials. Viscous fibers (guar, glucomannans, pectins, oat betaglucan and psyllium) continue to be seen as having hypocholesterolemic properties . Fiber physiological activity is thought to be critically dependent on its capacity to hydrate and thus increase the viscosity of human digest. American ginseng (AG) and Korean red ginseng (KRG) appear to affect glucose metabolism. AG seems to increase insulin secretion, while KRG has a blood glucose lowering effect through increasing insulin sensitivity. Corresponding author: Saša Magaš, MD, Merkur University Hospital, Vuk Vrhovac University Clinic for Diabetes, Endocrinology and Metabolic Diseases, Dugi dol 4a, HR-10000 Zagreb, Croatia e-mail: [email protected]


Ginsengs also show benefit in hypertension control by improving arterial stiffness. Salba is a variety of the ancient grain, used as food and remedy. It represents the highest whole food source of dietary fiber, omega-3 fatty acids, and is rich in vegetable protein, calcium, magnesium and iron, with a high total antioxidant capacity.

INTRODUCTION Diabetes mellitus is a chronic condition that occurs when the body cannot produce enough or effectively use insulin. Compared with individuals without diabetes, patients with type 2 diabetes mellitus (T2DM) have a considerably higher risk of cardiovascular morbidity and mortality, and are disproportionately affected by cardiovascular disease. Most of this excess risk is associated with the augmented prevalence of the well-known risk factors such as hypertension, dyslipidemia and obesity. Cardiovascular disease is a major burden in T2DM patients, emphasizing the importance of concurrent glucose management and reduction of disease-associated risk factors. Only 36% of T2DM patients are achieving goals for blood pressure (BP), 57% for LDL-cholesterol (LDL-C) and 50% for HbA1c targets, with only 13% of patients

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Saša Magaš, Lea Smirčić Duvnjak, Jelena Miočić / BENEFICIAL EFFECTS OF VISCOUS FIBER BLEND, AMERICAN AND RED KOREAN GINSENGS, SALVIA HISPANICA L. (SALBA, CHIA), OILY GRAIN, ON CARDIOVASCULAR RISK FACTOR MANAGEMENT IN TYPE 2 DIABETIC PATIENTS

meeting all three goals (DM-SCAN survey data). Cardiovascular disease causes one-third of deaths in Canada, more than any other illness (1). Although diet and lifestyle play critical roles in primary prevention, dietary approaches are viewed by many as ineffective in its present form. Therefore, any dietary approach and/or intervention that can reduce the risk of cardiovascular disease raise attention. Previous studies of individual interventions with viscous fiber blend, ginseng, and oily grain (Salvia hispanica L., salba) have repeatedly demonstrated them to attenuate glycemia and major and emerging cardiovascular disease risk factors in acute and longterm randomized-controlled trials.

VISCOUS FIBER BLEND

syndrome components (blood pressure, tryglycerides, HDL-C, fasting plasma glucose), total and LDL-C, C-reactive protein and endothelin-1 did not change in either group. In patients with T2DM and metabolic syndrome, the addition of guar gum to the usual diet improved cardiovascular and metabolic profiles by reducing HbA1c and trans-fatty acids (3). Slightly worse results in lowering the metabolic syndrome components in this study probably were due to using just one fiber (guar gum). Viscous fiber blend seems to be more effective. Over the past two decades, purified konjac flour, commonly known as konjac glucomannan (KGM), has been introduced on a relatively small scale into the United States and Europe, both as a food additive and dietary supplement (4). Clinical studies demonstrated that supplementing the diet with KGM significantly lowered plasma cholesterol and improved carbohydrate metabolism, bowel movement and colonic ecology (4).

Viscous fibers such as guar, glucomannans, pectins, oat betaglucan and psyllium continue to be seen as having hypocholesterolemic properties. The welldocumented lipid-lowering effects of fiber may be related to its viscosity, a phenomenon that has been understudied. When fiber is given against the background of a typical North American diet, benefits are well established, demonstrating modest but consistent effects on blood glucose and cholesterol levels (2). A Canadian study conducted in 2011 compared high-viscosity fiber against low-viscosity fiber in lowering LDL-C showed that despite the smaller quantity consumed, the high-viscosity fiber lowered LDL-C to a greater extent than low-viscosity fibers (2).

Animal model studies show benefits of using viscous fiber in lowering metabolic risk factors. In rats with streptozotocin induced diabetes, the guar gum diet significantly decreased serum concentrations of cholesterol, triacylglycerols and LDL-C, as well as atherogenic index. The most significant result in this study was the reduction of blood glucose in diabetic rats treated with the guar gum diet after 28 days versus non- and glibenclamide-treated rats. The gum promoted general improvement in the condition of diabetic rats in body weight and food intake in comparison with untreated rats (5).

Forty-four patients with T2DM and metabolic syndrome underwent a randomized controlled clinical trial conducted in 2013 (3). All patients followed their usual diet and the intervention group (n=23) received an additional 10 g/day of guar gum. In the intervention group, waist circumference (WC), glycated Hb (HbA1c), 24 h urinary albumin excretion and serum trans-fatty acids were reduced in comparison with baseline after 4 and 6 weeks (3). The only change in the control group was weight reduction. Other metabolic

Other animal studies also suggested that glucomannan itself and/or in conjunction with spirulina displayed hypolipemic and antioxidant effects when incorporated in functional ingredients. A study conducted in 2015 aimed to determine whether glucomannan-enriched or glucomannan plus spirulina-enriched squid-surimi improved plasma glucose and insulin levels in Zucker Fa/Fa rats fed a high saturated fat diet. Both glucomannan diets were able to reduce hyperglycemia and increase adipose

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tissue adiponectin levels in FA/FA rats, suggesting an anti-hypertrophic and insulin-sensitizing adipokine effect in this tissue (6). The magnitude of fiber physiological activity is thought to be critically dependent on its capacity to hydrate and thus increase the viscosity of human digesta. Fiber hydration affects the kinetics of nutrient bioavailability, mainly by slowing the rate of nutrient absorption in the small intestine and stimulating the rate of colonic fermentation, both of which may contribute to the reduction of blood glucose and lipid concentrations. These data support the inclusion of highviscosity fiber in the diet to reduce plasma lipids among apparently healthy individuals consuming a typical western diet.

GINSENGS American ginseng (AG) and Korean red ginseng (an Rg3-ginsenoside enriched extract; KRG) are two of the most commonly consumed ginsengs that have emerged as a promising complementary treatment in the management of T2DM patients. AG and KRG appear to affect glucose metabolism by separate but complementary mechanisms of action. While AG seems to increase insulin secretion, KRG might have a comparable blood glucose lowering effect through increasing insulin sensitivity. Different ethanol-extraction preparations of AG and KRG show no difference in attenuating postprandial glucose levels. Measures of insulin sensitivity index showed increased insulin sensitivity with KRG (30%) and AG (50%) extracts compared with placebo (p<0.05) (7). There is a potential for AG and KRG extracts to modulate insulin sensitivity (7). In a study conducted in 2000 by the Canadian authors, AG (Panax quinquefolius L.) reduced postprandial glycemia in nondiabetic subjects and subjects with T2DM. In nondiabetic subjects, no differences were found in postprandial glycemia between placebo


and ginseng when administered together with the glucose challenge. When ginseng was taken 40 minutes before the glucose challenge, significant reductions were observed (p<0.05) (8). American ginseng also shows additional benefit in hypertension control, by improving arterial stiffness (9). A total of 64 individuals with well-controlled essential hypertension and T2DM underwent a study in 2013 and completed the study. Compared to placebo, the group on AG showed significant reduction in radial augmentation index (arterial stiffness) by 5.3% (p=0.041) and systolic blood pressure by 11.7% (p<0.001) at 12 weeks. No effect was observed on diastolic blood pressure (9). There is doubt about the effectiveness of KRG in improving arterial stiffness. Results reported from the Korean and Canadian studies are quite opposite. In the Korean study conducted in 2011, 30 subjects in the active group (AG, KRG 3 g/day) and 34 subjects in the placebo group completed 3 months of treatment and then a per-protocol analysis was done. Systolic blood pressure and diastolic blood pressure at baseline, and at 1, 2, and 3 months were not different between the treated and placebo group (p>0.05). Analysis after adjustment for age, time-dependent mean arterial blood pressure, heart rate, and levels of fasting blood glucose and triglycerides showed no significant difference between the treated and placebo group; therefore, it was concluded that three-month treatment with KRG did not improve arterial stiffness in subjects with hypertension (10). On the other hand, the Canadian group of authors reported improved arterial stiffness following administration of 3 g/day of KRG. In this study, a total of 17 healthy fasted individuals underwent treatments consisting of 3 g/day of either placebo, KRG root, or a KRG root bioequivalent dose of ginsenoside or polysaccharide fractions. Compared to placebo, 3 g of KRG significantly lowered radial augmentation index by 4.6% (p=0.045), whereas the ginsenoside fraction comparably decreased radial augmentation index by 4.8% (p=0.057) and no effect was observed for polysaccharides (11).

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Regarding the safety of supplementation with the AG interventional material as an adjunct to conventional therapy (diet and/or medications) in T2DM, after 12 weeks of supplementation with AG, safety outcomes (kidney function, and creatinine), liver function (aspartate aminotransferase and alanine aminotransferase), and hemostatic function (prothrombin time and international normalization ratio) were not compromised in a population of T2DM patients at a high risk of cardiovascular disease, demonstrating that safety is noteworthy, despite articles which have continuously warned of the possible adverse effects of ginseng consumption (12).

SALVIA HISPANICA L. (SALBA, CHIA), OILY GRAIN Salvia hispanica L., commonly known as chia, is an annual plant belonging to the Lamiaceae family. Originating from countries such as Guatemala, Mexico and Colombia, chia seed was used and consumed as a source of energy and incorporated into a number of diets of the Aztec civilization (13). The lipid content in chia seeds varies from 25% to 40%, with 60% of the total lipids made up of aminolevulinic acid (ALA) (n-3) and 20% of linoleic acid (n-6). It is a good substitute source of polyunsaturated fatty acids (PUFA) to fish and other seed oils (14). It also has a significant concentration of dietary fiber (33.9 g/100 g) and protein (17 g/100 g). Of total dietary fiber, the greatest fraction (53.45 g/100 g) comprises insoluble fiber, which plays a role in satiety and proper bowel function. Rich in magnesium and phenolic compounds (mainly quercetin and kaempferol), chia seed offers significant antioxidant capacity (13). Its calcium and potassium content suggests it may be helpful in controlling high blood pressure (13). Animal model studies concerning the effects of chia seed feeding on rat plasma indicated that serum triglycerides and low-density lipoprotein (LDL) were significantly decreased, whereas high-density lipoprotein (HDL) and ω-3 PUFA levels were increased (14).

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Human studies conducted in the past few years also demonstrated the benefits of using chia as human diet supplementation (15-17). It took only seven weeks of supplementation of 25 g/day of milled chia seeds to elevate ALA and eicosapantenoic acid. The result was in agreement with previous studies conducted in hens, rats, and rabbits (15). After two months of supplementation of 235 kcal beverage containing soy protein, nopal, chia seed, and oat, 67 metabolic syndrome subjects achieved body weight loss and reduction of triglyceride and blood sugar levels (16). A randomized double blind trial on 11 healthy subjects conducted in 2010 with 50 g white bread containing different percentage of chia seed found reduction in postprandial glycemia (17).

CONCLUSION Modulations of dietary approaches and/or interventions to reduce the risk of cardiovascular disease should attract researchers’ and clinicians’ attention. The studies investigating the effect the consumption of chia seed, ginseng and viscous fibers on cardiovascular risk have reported inconclusive results. Most trials studied individual effects of viscous fibers, ginsengs, or chia seeds on cardiovascular risk factors, with inconclusive results. In particular, trials that would concurrently utilize health benefits of diet/herb collection that combines viscous fiber blend, omega-3 rich oily grains (salba), AG and KRG are lacking. We emphasize the need of randomized, double-blind, placebo-controlled clinical trials in order to obtain more reliable results.


REFERENCES 1.

Genest J, McPherson R, Frohlich J, Anderson T, Campbell N, Carpentier A, Couture P, et al. 2009 Canadian Cardiovascular Society/Canadian guidelines for the diagnosis and treatment of dyslipidemia and prevention of cardiovascular disease in the adult – 2009 recommendations. Can J Cardiol. 2009 Oct;25(10):567-579.

7. De Souza LR, Jenkins AL, Jovanovski E, Rahelić D, Vuksan V. Ethanol extraction preparation of American ginseng (Panax quinquefolius L) and Korean red ginseng (Panaxginseng C.A. Meyer): differential effects on postprandial insulinemia in healthy individuals. J Ethnopharmacol. 2015 Jan 15;159:55-61.

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Vuksan V, Jenkins AL, Rogovik AL, Fairgrieve CD, Jovanovski E, Leiter LA. Viscosity rather than quantity of dietary fibre predicts cholesterollowering effect in healthy individuals. Br J Nutr. 2011 Nov;106(9):1349-1352.

8. Vuksan V, Sievenpiper JL, Koo VY, Francis T, Beljan-Zdravkovic U, Xu Z, Vidgen E. American ginseng (Panax quinquefolius L) reduces postprandial glycemia in nondiabetic subjects and subjects with type 2 diabetes mellitus. Arch Intern Med. 2000 Apr 10;160(7):1009-1013.

3. Dall’Alba V, Silva FM, Antonio JP, Steemburgo T, Royer CP, Almeida JC, Gross JL, Azevedo MJ. Improvement of the metabolic syndrome profile by soluble fibre – guar gum – in patients with type 2 diabetes: a randomised clinical trial. Br J Nutr. 2013 Nov 14;110(9):1601-1610. 4. Chua M, Baldwin TC, Hocking TJ, Chan K. Traditional uses and potential health benefits of Amorphophallus konjac K. Koch ex N.E. Br J Ethnopharmacol. 2010 Mar 24;128(2):268-278. 5. Samarghandian S, Hadjzadeh MA, Amin Nya F, Davoodi S. Antihyperglycemic and antihyperlipidemic effects of guar gum on streptozotocin-induced diabetes in male rats. Pharmacogn Mag. 2012 Jan;8(29):65-72. 6. Vázquez-Velasco M, González-Torres L, Méndez MT, Bastida S, Benedí J, González-Muñoz MJ, Sánchez-Muniz FJ. Glucomannan and Glucomannan plus spirulina-enriched squid surimi added to high saturated diet affect glycemia, plasma and adipose leptin and adiponectin levels in growing FA/FA rats. Nutr Hosp. 2015 Dec 1;32(6):2718-2724.


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Mucalo I, Jovanovski E, Rahelić D, Božikov V, Romić Z, Vuksan V. Effect of American ginseng (Panax quinquefolius L.) on arterial stiffness in subjects with type 2 diabetes and concomitant hypertension. J Ethnopharmacol. 2013 Oct 28;150(1):148-153.

10. Rhee MY, Kim YS, Bae JH, Nah DY, Kim YK, Lee MM, Kim HY. Effect of Korean red ginseng on arterial stiffness in subjects with hypertension. J Altern Complement Med. 2011 Jan;17(1):45-49. 11. Jovanovski E, Jenkins A, Dias AG, Peeva V, Sievenpiper J, Arnason JT, Rahelic D, Josse RG, Vuksan V. Effects of Korean red ginseng (Panax ginseng C.A. Mayer) and its isolated ginsenosides and polysaccharides on arterial stiffness in healthy individuals. Am J Hypertens. 2010 May;23(5): 469-472. 12. Mucalo I, Jovanovski E, Vuksan V, Božikov V, Romić Z, Rahelić D. American ginseng extract (Panax quinquefolius L.) is safe in long-term use in type 2 diabetic patients. Evid Based Complement Alternat Med. 2014;2014:969168.

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13. de Souza Ferreira C, dd Sousa Fomes Lde F, da Silva GE, Rosa G. Effect of chia seed (Salvia hispanica L.) consumption on cardiovascular risk factors in humans: a systematic review. Nutr Hosp. 2015 Nov 1;32(5):1909-1918.

16. Martha GC, Armando RT, Carlos AA, et al. A dietary pattern including Nopal, chia seed, soy protein, and oat reduces serum triglycerides and glucose intolerance in patients with metabolic syndrome. J Nutr. 2012;142(1):64-69

14. Mohd Ali N, Yeap SK, Ho WY, Beh BK, Tan SW, Tan SG. The promising future of chia, Salvia hispanica L. J Biomed Biotechnol. 2012; 2012:171956.

17. Vuksan V, Jenkins AL, Dias AG, et al. Reduction in postprandial glucose excursion and prolongation of satiety: possible explanation of the long-term effects of whole grain salba (Salvia hispanica L.). Eur J Clin Nutr. 2010;64(4):436-438.

15. Jin F, Nieman DC, Sha W, et al. Supplementation of milled chia seeds increases plasma ALA and EPA in postmenopausal women. Plant Foods Human Nutr. 2010;67:105-110.

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Review

Endocrine and Metabolic Institute, Department of Endocrinology and Metabolism, Cleveland Clinic, Cleveland, Ohio, USA

MANAGEMENT OF THYROID NODULES Mario Škugor

Key words: thyroid nodule, ultrasound features, fine needle aspiration, Bethesda system, thyroidectomy

SUMMARY Thyroid nodules are very common, but only a minor proportion of these are of clinical significance. However, what is most important, some are malignant. Current guidelines recommend evaluation of cold thyroid nodules that are 10 mm or larger in size. Fine needle aspiration (FNA) is the preferred method for evaluation of the nature of the lesion. Results should be reported according to the Bethesda System for Reporting Thyroid Cytopathology. Most nodules (55%-75%) will be classified as benign and a minority (2%-5%) will be malignant. The rest of FNA specimens are reported as either inadequate or inconclusive. In the cases of inadequate specimen, FNA should be repeated under ultrasound guidance, which will result in adequate sample in 60%-80% of cases. Clinical Corresponding author: Mario Škugor, MD, FACE, Endocrine and Metabolic Institute, Department of Endocrinology and Metabolism, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio, USA E-mail: [email protected]


decision regarding management of the nodules with inconclusive FNA results should be guided by the personal and family history, as well as by the presence or absence of the worrisome ultrasound features. Genetic analysis of mutations in FNA specimens has been reported to be helpful in some situations, but its role in the management of thyroid nodules is evolving and currently is not quite clear.

INTRODUCTION Thyroid nodules are found by palpation in about 5% of females and 1% of males (1) in iodine sufficient areas. Ultrasound examination of thyroid gland reveals much more nodules, especially in females and in the elderly. Incidence as high as 68% is reported using the high frequency probe (2). Only a minority of these nodules are of clinical significance. Some are of cosmetic concern, others are causing mass related symptoms, and most importantly, some are malignant. Malignancy rate has been reported to be about 9% with clinically non-apparent solitary nodules, and 6%7% of those that are part of multinodular goiters (3). More than 90% of thyroid carcinomas are considered well differentiated and most of them are papillary thyroid carcinomas, followed by follicular thyroid

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Mario Škugor / MANAGEMENT OF THYROID NODULES

carcinomas. Thyroid cancer rates increased between 1973 and 2002 worldwide, with the mean increase of 48.0% for males and 66.7% for females (4). It is predicted that papillary thyroid carcinoma will become the third most common cancer in women in the USA by 2019 (5).

THYROID NODULE EVALUATION Thyroid nodules can be detected by inspection and palpation, but more often they are detected incidentally on imaging studies done for other reasons, most commonly while performing carotid ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI) of the head, neck and thorax, as well as whole body positron emission tomography (PET) scans. Nodules may be solitary or part of multinodular thyroid gland. Details obtained by reviewing the family and personal history, as well as physical examination will help delineate the risk of malignancy in the nodule. Important aspects of the history that increase the risk of malignancy include prior radiation therapy to the head and neck, rapid growth of the nodule, and presence of the mass related symptoms, such are dysphagia, and dysphonia. An increased risk is also seen in male sex and extremes of age (<20 years or >70 years) with a family history of thyroid carcinoma in first degree relative and family history of several genetic syndromes (MEN-2, Cowden syndrome Carney complex, Pendred syndrome, Werner syndrome and familial adenomatous polyposis) (6). Another important factor is the length of time that the nodule is present. A clinically stable nodule present for years is unlikely to be malignant. Physical examination should be focused on the possible signs of hyperthyroidism, size of the gland and nodule, consistency and movability of the masses in the thyroid bed, and presence or absence of neck lymphadenopathy. Ultrasound is the imaging technique used for initial assessment of the gland and thyroid nodules. It is noninvasive, inexpensive, quick, and offers excellent

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spatial resolution. Examination of the entire thyroid gland and surrounding neck for abnormal masses or lymphadenopathy is mandatory. Ultrasound report should contain information on the size of the thyroid gland, appearance of the parenchyma (homogeneous or heterogeneous), number and location of thyroid nodules. Each clinically significant nodule should be measured in three dimensions, and include information on the composition (solid, is there any cystic component, spongiform nature), echogenicity (hypoechoic, isoechoic or hyperechoic in comparison with normal thyroid parenchyma), shape, appearance of the margin, presence and type of calcifications (complete eggshell, incomplete eggshell, rough calcifications or microcalcifications), and information on vascularity. Finally, the presence or absence of suspect central and lateral neck lymph nodes should be noted and if present described in terms of size, shape, vascularity and echogenicity. There is considerable literature on the use of US elastography of thyroid nodules (measurement of tissue stiffness) in thyroid malignancy risk assessment. This technique requires specialized software (sold separately from the ultrasound machine). Initial study of 92 patients suggested positive and negative predictive values of near 100% (7). However, subsequent studies had considerably different results. The largest study in 706 patients obtained a positive predictive value of only 36% (8). Also, this technique can only be applied to solid nodules and is highly operator dependent. At this time, ultrasound elastography cannot be recommended for routine use. The American Thyroid Association recommends further evaluation by fine needle aspiration (FNA) biopsy of most nodules that are larger than 10 mm because of a higher likelihood of carrying clinically significant malignancy (9). Exceptionally, smaller nodules require evaluation because of ultrasound features, presence of clinical symptoms, or because of suspect neck lymphadenopathy. The incidence of carcinoma in pure cysts is negligible and it is


acceptable to monitor these using US regardless of size. Spongiform nodules have a very low risk of harboring carcinoma and could be evaluated with FNA when 2 cm or larger in size, but observation is also an acceptable option (9).

ULTRASOUND STRATIFICATION OF THE RISK OF MALIGNANCY Ultrasound features are helpful in stratification of the malignancy risk and are used to classify nodules in the following groups: High suspicion – estimated risk of 70%-90%; solid (can contain small cystic portion), hypoechoic nodule with one or more of the following characteristics: microcalcifications, irregular margins, extrathyroidal extension, incomplete rim (egg-shell) calcifications with extrusive soft tissue component, and taller than wide shape. It is acceptable to perform FNA even if nodules are smaller than 1 cm, but in general 1 cm is acceptable size for FNA even in these cases. Intermediate suspicion – estimated risk of 10%20%; solid (can contain cystic portion), hypoechoic nodule with smooth border and without other high suspicion features. FNA should be performed when the nodule is 1 cm or larger. Low suspicion – estimated risk of 5%-10%; solid isoechoic or hyperechoic nodule, or partially cystic nodule with eccentric solid component and without high suspicion features. FNA should performed when the nodule is 1.5 cm or larger in size. Very low suspicion – estimated risk <3%; spongiform nodule or predominantly cystic nodule without high suspicion features. FNA should be considered at a size of 2 cm or larger, although observation with ultrasound is an acceptable option. Benign – estimated risk <1%; pure cysts. No FNA is necessary.


If US examination indicates a need for FNA, biochemical tests of thyroid function should be obtained before FNA is undertaken. Thyroid-stimulating hormone (TSH) measurement is sufficient unless there is clinical suspicion of hyperthyroidism. If so, then the levels of thyroxine (T4) and triiodothyronine (T3) should also be determined. There is no reason for measurement of thyroglobulin level prior to FNA. If TSH is less than the lower level of the reference range, radioactive iodine scan should be performed to determine if the nodule is hyperactive (hot nodule), isoactive (warm nodule) or hypoactive (cold nodule). Hot nodule very rarely contains malignancy and FNA biopsy is not necessary (10). Cold nodules, as well as nodules associated with normal or elevated TSH should undergo FNA biopsy. Elevated TSH, even in the upper part of the reference range, is associated with an increased risk of thyroid carcinoma when thyroid nodule is present (11). The FNA is a cost effective and most accurate method for determination of the nature of the thyroid nodule. The best technique to perform FNA is not clear. The size of the needle varies at gauge 27-21. We prefer a longer needle (37 mm), especially for posteriorly located nodules. The number of needle passes varies from 2 to 4 per nodule. We prefer 2 passes. The number of cytologic slides prepared varies between 3 and 6. We prefer 3-4 slides. US guidance of FNA is reported to decrease the rate of non-diagnostic and false-negative FNAs (12, 13). It is recommended to use US guidance in all nodules that have a significant cystic component (>25%) or are difficult to palpate (smaller nodules or those that are located posteriorly in thyroid gland). Results obtained by FNA cytology should be reported using the diagnostic groups defined in the Bethesda System for Reporting Thyroid Cytopathology (14, 15). This system recognizes six categories of findings and provides carcinoma risk estimates in each category based on review of literature reports and expert opinion.

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Categories are as follows: Nondiagnostic or unsatisfactory – actual risk of malignancy in excised lesions ranges from 9% to 32%, average 20% (16). Only a minority of lesions in this category are surgically removed and the estimated risk of malignancy in all lesions in this category by the Bethesda system is 1%-4% (17). Benign, estimated risk is 0-3% (17) – actual risk is reported to be 1%-10% in excised nodules with average of 2.5% (16). Atypia of undetermined significance or follicular lesion of undetermined significance – estimated risk is 5%-15% (17) and reported actual risk 6%48%, average 14% (16). Follicular neoplasm or suspect of follicular neoplasm – estimated risk is 15%-30% (17) and reported risk 14%-34%, average 25% (16). Suspect of malignancy – estimated risk 60%-75% (17) and reported risk 53%-97%, average 70% (16). Malignant – estimated risk is 97%-99% (17) with reported risk of 94%-100%, average 99% (16). Nondiagnostic or unsatisfactory FNA samples are those that fail to meet quantitative or qualitative criteria for cytologic adequacy. For a sample to be adequate, it has to contain at least six groups of at least ten well preserved and visualized follicular cells, preferably on the single slide (18). Exception to this rule is a sample containing abundant colloid and few well visualized follicular cells that could be considered benign despite failing to meet the above criteria for adequacy (7). When Bethesda classification is applied to large series of patients, the majority (89%-95%) of samples are considered adequate for interpretation. If the initial sample is not satisfactory, FNA should be repeated with US guidance and, if possible, on site cytologic evaluation. Some data suggest that wait period of 3 months is necessary to avoid false-positive findings

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due to reactive/reparative changes (19). Other data suggest that timing of repeat FNA does not affect the results (20). Our practice is to repeat FNA in 2-4 weeks. Repeat FNA results in a specimen that is adequate for analysis in 60%-80% of cases (21, 22). If the cytologic diagnosis is inconclusive on repeat FNA, it is reasonable to closely follow-up by US examination those nodules without the high risk of US features. We follow them up at 3- to 4-month interval, then at 6 months, and then yearly for several years and recommend surgery if there is significant growth of the nodule defined as increase of >20% in two dimensions. For nodules with high risk US features, surgical intervention should be considered. Of the FNA samples adequate for analysis, 55%74% are considered benign and 2%-5% are malignant (16, 23). The rest of samples are not conclusive and include 2%-18% of samples read as atypia or follicular lesion of unknown significance, 2%-25% of follicular neoplasms, and 1%-6% suspect of thyroid carcinoma (16, 23-25). When a large, blinded prospective study was conducted to assess inter-observer concordance, it was found that the categories of suspect of carcinoma and atypia or follicular neoplasm of unknown significance produced the highest discordance rate, illustrating inherent limitations of the current system (26). Nevertheless, the Bethesda system has proved beneficial in allowing us to use the same terminology by different practitioners and better convey of the risk of malignancy. The rate of non-conclusive cytology findings varies between 2% and 16% in different series (16, 23); furthermore, 7%-26% of these nodule were resected and the malignancy rate reported was in range of 2%4% in all nodules, and 9%-32% in those resected. The US features are helpful in assessing the risk of malignancy and guide clinical decision when FNA result is inconclusive. Malignancy risk has been reported to be 25% when the nodule harbored microcalcifications, irregular margins, taller than wider shape or hypoechogenicity, while it was 4% when these features were lacking (27).


The use of core needle biopsy, and in recent years the use of molecular genetic markers, especially BRAF and RAS mutations, have been reported to offer useful information in classifying thyroid nodules with inconclusive FNA results (28-30). The spectrum of mutations implicated in the pathogenesis of differentiated thyroid carcinoma include genes for nuclear proteins (PAX-8-PPARγ), tyrosine kinase receptors (RET/PTC, NTRK) and signaling proteins (BRAF, RAS), and the list is growing rapidly. Rearrangements of RET/PTC are reported only in papillary thyroid carcinoma. The BRAF, RAS, or RET/PTC mutations are present in >70% of papillary thyroid carcinomas (31). The V600E mutation of BRAF is associated with aggressive forms of papillary thyroid carcinoma (32, 33). The RAS proto-oncogene mutations (HRAS, NRAS, KRAS), or PAX-8-PPARγ rearrangement are seen in nearly 70% of follicular thyroid carcinomas (34). One study found that the presence of BRAF mutation, RET/PTC, or PAX-8-PPARγ rearrangements in inconclusive FNA specimen had a 100% specificity for thyroid cancer, and that RAS mutation indicated 83%-87% of malignancy risk in any FNA specimen (35). However, the false-positive BRAF mutations are reported, and the RET/PTC rearrangements are found in benign conditions such as trabecular adenoma and Hashimoto thyroiditis (36, 37). Molecular markers could be used for diagnostic or prognostic purposes. However, in most clinical situations, the answer we are looking for is not if the lesion is malignant. The question is: is this lesion benign? A lesion classified as follicular neoplasm, the one containing atypical cells, or the one suspect of thyroid carcinoma is usually managed by surgery and confirmation of the carcinoma in such a lesion changes a little in the management (it does affect the extent of surgery in some cases). Confirmation of the benign nature of the lesion is, thus, much more important and should be the goal of genetic analysis. Using the panel of the 7 gene analysis (BRAF, HRAS, KRAS, NRAS, RET/PTC1, RET/PTC3, PAX8/PPARƴ) in several studies showed improved


sensitivity for cancer (35, 38), but more importantly, 94% of the nodules testing negative for these mutations were benign on postoperative histopathology (38). Another approach employing a gene expression classifier by using mRNA analysis of 167 genes (167 GEC) has been reported to yield excellent sensitivity of 90% but only 53% specificity for the diagnosis of thyroid carcinoma in FNA samples carrying designation of atypia of undetermined significance or follicular lesion of undetermined significance (39). At this time, the field of mutation analysis in FNA samples is evolving, analytical and clinical validity are being established, long term effects of its use are lacking, and it is still not clear where the best clinical utility of these tests lies. It is likely that some combination of mutational analysis will offer valuable clinical insight in the future. There is general consensus that nodules with benign FNA results do not require additional diagnostic or therapeutic intervention in immediate term. In several retrospective analyses, the malignancy rate in such nodules was 1%-2% (40-42). Clinical follow up if the nodule is visible or palpable and US follow up if it is not are appropriate. Significant increase in size (>20% in two dimensions) should prompt the consideration of surgery. There is some evidence that larger nodules have a higher FNA false-negative rate, especially those over 4 cm in size, but it is unclear if these nodules should be treated differently than the smaller ones. If the cytologic diagnosis of primary thyroid carcinoma is made, surgery is recommended for most patients. Close follow up could be offered to the individuals who have a high surgical risk because of comorbidities, patients with short life expectancy, patients whose other medical or surgical conditions need to be addressed before thyroid surgery, and patients with very low risk thyroid carcinomas (less than 10 mm in size, no evidence of local invasion or metastases, and no cytologic evidence of aggressive disease). Support for observation regarding very low

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risk thyroid carcinomas comes from studies performed in Japan demonstrating a very low rate of tumor enlargement (5% at 5 years and 8% at 10 years of follow up), and development of lymph node metastases (1.7% at 5 years and 3.8% at 10 years). Only 191 of 1235 patients required surgery during the follow up and only one had recurrence during the follow up ranging 1-20 years (43). Interestingly, this study also found the risk of clinical progression of thyroid carcinoma to be 8.9% in those younger than 40, 3.5% in those aged 40-60 and 1.9% in those older than 60 years. Another study followed 230 patients for average of 5 years and observed a 7% rate of nodal enlargement and 1% rate of lymph node metastasis development; 7% underwent surgery and no recurrence was observed after 1- to 12year follow up, suggesting no harm from delaying the

surgery (44). These observations are intriguing, but have to be confirmed in other centers and populations.

CONCLUSION The currently advocated system for evaluation and management of thyroid nodules is well defined and easy to follow. However, the system is far from being perfect and we still have difficulties recognizing the aggressive thyroid malignancies in their early stages, as well as recognizing those that will behave indolently and require less aggressive treatment. Further research is necessary to improve our understanding of these disorders and answers are likely to be found in the molecular analysis (genetic, epigenetic and proteomic) of these lesions.

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