Modulation of diabetes-related liver injury by the HMGB1/TLR4 inflammatory pathway.

Chronic inflammation plays an essential role in the development of diabetic complications. Understanding the molecular mechanisms that support inflammation is a prerequisite for the design of novel anti-inflammatory therapies. These would take into consideration circulating levels of cytokines and damage-associated molecular patterns (DAMPs) that include the high mobility group box 1 (HMGB1) protein which, in part, promotes the inflammatory response through TLR4 signaling. The liver, as the source of circulating cytokines and acute-phase proteins, contributes to the control of systemic inflammation. We previously found that liver injury in streptozotocin-induced diabetic rats correlated with the level of oxidative stress, increased expression of HMGB1, and with the activation of TLR4-mediated cell death pathways. In the present work, we examined the effects of ethyl pyruvate (EP), an inhibitor of HMGB1 release/expression, on the modulation of activation of the HMGB1/TLR4 inflammatory cascade in diabetic liver. We observed that increased expression of inflammatory markers, TNF-α, IL-6, and haptoglobin in diabetic liver was associated with increased HMGB1/TLR4 interaction, activation of MAPK (p38, ERK, JNK)/NF-κB p65 and JAK1/STAT3 signaling pathways, and with decreased expression of Nrf2-regulated antioxidative enzymes. The reduction in HMGB1 expression as the result of EP administration reduced the pro-inflammatory activity of HMGB1 and exerted a protective effect on diabetic liver, which was observed as improved liver histology and antioxidant and inflammatory statuses. Our results suggest that prevention of HMGB1 release and blockage of the HMGB/TLR4 axis represents a potentially effective therapeutic strategy aimed at ameliorating diabetes-induced inflammation and ensuing liver injury.

Oxidative stress-dependent contribution of HMGB1 to the interplay between apoptosis and autophagy in diabetic rat liver.

The progression of oxidative stress, resulting cell damage, and cell death underlies the etiology of liver damage/dysfunction as a complication of diabetes. High-mobility group box 1 (HMGB1) protein, a chromatin-binding nuclear protein and damage-associated molecular pattern molecule, is integral to oxidative stress and signaling pathways regulating cell death and cell survival. We previously found that in streptozotocin (STZ)-induced diabetic rats, reduction of oxidative stress after melatonin administration lowered necrotic cell death and increased expression of HMGB1 and hepatocellular damage. In the present study, we examined whether alleviation of diabetes-attendant oxidative stress and ensuing change in HMGB1 expression influence the dynamic equilibrium between apoptosis/autophagy and liver damage. We observed that elevated HMGB1 protein levels in diabetic rat liver accompanied increased interactions of HMGB1 with TLR4 and RAGE, and activation of the intrinsic apoptotic pathway and Beclin 1-dependent autophagy. The absence of p62 degradation in diabetic rat liver pointed to defective autophagy which was responsible for lower autophagosome/autophagolysosome formation and an increased apoptosis/autophagy ratio. Compared to diabetic rats, in melatonin-treated diabetic rats, the structure of liver cells was preserved, HMGB1/TLR4 interaction and downstream apoptotic signaling were significantly reduced, HMGB1/Beclin 1 colocalization and interactions were augmented and Beclin 1-mediated autophagy, mithophagy in particular, were increased. We concluded that in mild oxidative stress, HMGB1 is cytoprotective, whereas in intense oxidative stress, HMGB1 actions promote cell death and liver damage. Since reduced HMGB1 binds to RAGE but not to TLR4, redox modification of HMGB1 as a mechanism regulating the cross-talk between apoptosis and autophagy in diabetes is discussed.

Catalase inhibition in diabetic rats potentiates DNA damage and apoptotic cell death setting the stage for cardiomyopathy.

Diabetes is a risk factor for cardiovascular disease that has a multifactorial etiology, with oxidative stress as an important component. Our previous observation of a significant diabetes-related increase in rat cardiac catalase (CAT) activity suggested that CAT could play a major role in delaying the development of diabetic cardiomyopathy. Thus, in the present work, we examined the effects of the daily administration of the CAT inhibitor, 3-amino-1,2,4-triazole (1 mg/g), on the hearts of streptozotocin (STZ)-induced diabetic rats. Administration of CAT inhibitor was started from the 15th day after the last STZ treatment (40 mg/kg/5 days), and maintained until the end of the 4th or 6th weeks of diabetes. Compared to untreated diabetic rats, at the end of the observation period, CAT inhibition lowered the induced level of cardiac CAT activity to the basal level and decreased CAT protein expression, mediated through a decline in the nuclear factor erythroid-derived 2-like 2 /nuclear factor-kappa B p65 (Nrf2/NF-κB p65) subunit ratio. The perturbed antioxidant defenses resulting from CAT inhibition promoted increased H2O2production (P < 0.05) and lipid peroxidation (P < 0.05). Generated cytotoxic stimuli increased DNA damage (P < 0.05) and activated pro-apoptotic events, observed as a decrease (P < 0.05) in the ratio of the apoptosis regulator proteins Bcl-2/Bax, increased (P < 0.05) presence of the poly(ADP-ribose) polymerase-1 (PARP-1) 85 kDa apoptotic fragment and cytoplasmic levels of cytochrome C. These findings confirm an important function of CAT in the suppression of events leading to diabetes-promoted cardiac dysfunction and cardiomyopathy.

Hepatoprotective effects of melatonin against pronecrotic cellular events in streptozotocin-induced diabetic rats.

Oxidative stress-mediated damage to liver tissue underlies the pathological alterations in liver morphology and function that are observed in diabetes. We examined the effects of the antioxidant action of melatonin against necrosis-inducing DNA damage in hepatocytes of streptozotocin (STZ)-induced diabetic rats. Daily administration of melatonin (0.2 mg/kg) was initiated 3 days before diabetes induction and maintained for 4 weeks. Melatonin-treated diabetic rats exhibited improved markers of liver injury (P < 0.05), alkaline phosphatase, and alanine and aspartate aminotransferases. Melatonin prevented the diabetes-related morphological deterioration of hepatocytes, DNA damage (P < 0.05), and hepatocellular necrosis. The improvement was due to containment of the pronecrotic oxygen radical load, observed as inhibition (P < 0.05) of the diabetes-induced rise in lipid peroxidation and hydrogen peroxide increase in the liver. This was accompanied by improved necrotic markers of cellular damage: a significant reduction in cleavage of the DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP-1) into necrotic 55- and 62-kDa fragments, and inhibition of nucleus-to-cytoplasm translocation and accumulation in the serum of the high-mobility group box 1 (HMGB1) protein. We conclude that melatonin is hepatoprotective in diabetes. It reduces extensive DNA damage and resulting necrotic processes. Melatonin application could thus present a viable therapeutic option in the management of diabetes-induced liver injury.

Activation level of JNK and Akt/ERK signaling pathways determinates extent of DNA damage in the liver of diabetic rats.

AIMS: Diabetes-related oxidative stress conditions lead to progressive tissue damage and disfunctionality. Mechanisms underlying liver pathophysiology during diabetes are not fully understood. The aim of this study was to find relationship between diabetes-related DNA damage in the rat liver and activities of prosurvival signaling pathways. METHODS: Effect of diabetes was analyzed two (development stage) and eight weeks (stable diabetes) after single intraperitoneal injection of streptozotocin. Extent of DNA damage, analysed by comet assay, was corelated with oxidative status (plasma level of ROS, liver antioxidant capacity) and activity/abundance of kinases (Akt, p38, ERK1, JNK, JAK) and transcription factors NF-κB p65 and STAT3. RESULTS: Significant DNA damage in development stage is accompanied by elevated plasma levels of O(2)(-) and H(2)O(2), decreased activities of CAT, MnSOD, and GST in the liver and increased activation of proapoptotic JNK signal pathway. Lower DNA damage in stable diabetes, is accompanied by elevated plasma level of O(2)(-), restored antioxidative liver enzyme activity, decreased activation of JNK and increased activation of prosurvival Akt and ERK signal pathways. CONCLUSION: These findings indicate that level of DNA damage in diabetic liver depends on the extent of oxidative stress, antioxidant activity and balance between JNK and Akt/ERK signal pathways activation .

STAT3/NF-κB interactions determine the level of haptoglobin expression in male rats exposed to dietary restriction and/or acute phase stimuli.

Haptoglobin is a constitutively expressed protein which is predominantly synthesized in the liver. During the acute-phase (AP) response haptoglobin is upregulated along with other AP proteins. Its upregulation during the AP response is mediated by cis-trans interactions between the hormone-responsive element (HRE) residing in the haptoglobin gene and inducible transcription factors STAT3 and C/EBP ß. In male rats that have been subjected to chronic 50% dietary restriction (DR), the basal haptoglobin serum level is decreased. The aim of this study was to characterize the trans-acting factor(s) responsible for the reduction of haptoglobin expression in male rats subjected to 50% DR for 6 weeks. Protein-DNA interactions between C/EBP and STAT families of transcription factors and the HRE region of the haptoglobin gene were examined in livers of male rats subjected to DR, as well as during the AP response that was induced by turpentine administration. In DR rats, we observed associations between the HRE and C/EBPα/ß, STAT5b and NF-κB p50, and the absence of interactions between STAT3 and NF-kB p65. Subsequent induction of the AP response in DR rats by turpentine administration elicited a normal, almost 2-fold increase in the serum haptoglobin level that was accompanied by HRE-binding of C/EBPß, STAT3/5b and NF-kB p65/p50, and the establishment of interaction between STAT3 and NF-κB p65. These results suggest that STAT3 and NF-κB p65 crosstalk plays a central role while C/EBPß acquires an accessory role in establishing the level of haptoglobin gene expression in male rats exposed to DR and AP stimuli.

The effect of chronic food restriction on immunopositive ACTH cells in peripubertal female rats.

In peripubertal female rats, we have previously found that 50% food restriction (FR) increases plasma IL-6, haptoglobin and both alanine transaminase (ALT) and alkaline phosphatase (AST) aminotransferases, indicating the existence of an inflammatory response. To study whether such FR influences the hypothalamic-pituitary-adrenal (HPA) axis, we examined by immunohistochemistry the morphofunctional features of pituitary adrenocorticotroppic (ACTH) cells. In FR rats the volume and volume density of ACTH cells as well as plasma ACTH levels were increased by 17.6%, 12.5% and 13.4%, respectively, in comparison with controls (p<0.05). We concluded that chronic FR is a systemic stressor in young females, capable to stimulate the HPA axis, probably as a result of IL-6 action.

The absence of cardiomyopathy is accompanied by increased activities of CAT, MnSOD and GST in long-term diabetes in rats.

The activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST), the incidence of DNA damage, the activation of poly (ADP-ribose) polymerase-1 (PARP-1), a marker of DNA repair, and connective tissue growth factor (CTGF), a marker of tissue fibrosis, were examined in the hearts of rats for 16 weeks after diabetes induction by streptozotocin (STZ) administration. A 150% increase in CAT activity was detected at the end of the 2nd week post-STZ administration, and CAT activity remained 80% above the control level throughout 16 weeks. While total SOD and CuZn-SOD exhibited progressively decreasing activities, those of Mn-SOD and GST were elevated. Neither DNA strand breaks (apoptosis or necrosis) nor changes in PARP-1 activity and in CTGF levels (fibrosis) were observed in the diabetic heart. The absence of cardiomyopathy is accompanied with increased activities of CAT, MnSOD and GST.

Association of the glucocorticoid receptor with STAT3, C/EBPbeta, and the hormone-responsive element within the rat haptoglobin gene promoter during the acute phase response.

Upregulation of haptoglobin (Hp) expression in the rat during the acute phase (AP) response is the result of synergistic effects of IL-6-, IL-1beta-, and corticosterone-activated signaling pathways. IL-6 signaling terminates in cis-trans interactions of the Hp gene hormone-responsive element (HRE) with transcription factors STAT3 and C/EBPbeta. The aim of this study was to examine the unresolved molecular mechanism of glucocorticoid action. A 3-fold rise in serum corticosterone at 2 and 4 h of the AP response induced by turpentine administration preceded a 2.3-fold increase in the rate of Hp gene transcription at 12 h that was accompanied by a 4.8-fold increase in glucocorticoid receptor (GR), the appearance of an 86-kDa STAT3 isoform and 3.9-, 1.9-, and 1.7-fold increased amounts of 91-kDa STAT3, 35- and 42-kDa C/EBPbeta isoforms in the nucleus. These events resulted in 4.6- and 2.5-fold increased Hp levels in the liver and serum at 24 h. HRE affinity chromatography and immunoblot analysis revealed that maximal occupancy of the HRE with GR, STAT3, and C/EBPbeta at 12 h correlated with increased transcriptional activity of the Hp gene. Coimmunoprecipitation experiments showed that activated GR established de novo interaction with STAT3 isoforms while GR-C/EBPbeta interactions observed during basal transcription increased during the AP response. Computer analysis of the HRE disclosed two potential GR-binding sites: one overlapping STAT3, another adjacent to a C/EBPbeta-binding site. This finding and the experimental results suggest that activated GR through direct interactions with STAT3 and C/EBPbeta, participates in Hp gene upregulation as a transcriptional coactivator.

The radioprotective effect of alpha2-macroglobulin: a morphological study of rat liver.

BACKGROUND: Administration of the acute-phase protein alpha2-macroglobulin (MG) prior to total-body irradiation of rats with a 6.7 Gy (LD50) dose of X-rays exerts a radioprotective effect. MATERIAL/METHODS: MG was administered 30 min before irradiation with a 6.7 Gy (LD50) dose of X-rays. Its radioprotective efficacy was compared with that of the synthetic agent amifostine (WR-2721), a sulfhydryl compound which is currently the most effective radioprotector in clinical use. After administration of either MG or amifostine, changes in body and liver weight were recorded and histological liver sections were examined during a four-week follow-up period. RESULTS: As observed in the experimental group administered amifostine, rats that received MG prior to irradiation exhibited 100% survival and restoration of the body and liver weight to the control values. The morphological damage seen in the liver after irradiation of untreated rats was absent in both the MG- and amifostine-pretreated rats. Also, hepatocytes and granulated cells had prominent nuclei and did not exhibit major changes in volume. Dilation of the central vein was not observed. CONCLUSIONS: Administration of MG before irradiation, similar to pretreatment with amifostine, provided complete survival of experimental rats and recovery of liver weight and preserved major histological parameters of the liver.