The Zinc Transporter Slc30a8/ZnT8 Is Required in a Subpopulation of Pancreatic α-Cells for Hypoglycemia-induced Glucagon Secretion

SLC30A8 encodes a zinc transporter ZnT8 largely restricted to pancreatic islet β- and α-cells, and responsible for zinc accumulation into secretory granules. Although common SLC30A8 variants, believed to reduce ZnT8 activity, increase type 2 diabetes risk in humans, rare inactivating mutations are protective. To investigate the role of Slc30a8 in the control of glucagon secretion, Slc30a8 was inactivated selectively in α-cells by crossing mice with alleles floxed at exon 1 to animals expressing Cre recombinase under the pre-proglucagon promoter. Further crossing to Rosa26:tdRFP mice, and sorting of RFP⁺: glucagon⁺ cells from KO mice, revealed recombination in ∼30% of α-cells, of which ∼50% were ZnT8-negative (14 ± 1.8% of all α-cells). Although glucose and insulin tolerance were normal, female αZnT8KO mice required lower glucose infusion rates during hypoglycemic clamps and displayed enhanced glucagon release (p < 0.001) versus WT mice. Correspondingly, islets isolated from αZnT8KO mice secreted more glucagon at 1 mm glucose, but not 17 mm glucose, than WT controls (n = 5; p = 0.008). Although the expression of other ZnT family members was unchanged, cytoplasmic (n = 4 mice per genotype; p < 0.0001) and granular (n = 3, p < 0.01) free Zn²⁺ levels were significantly lower in KO α-cells versus control cells. In response to low glucose, the amplitude and frequency of intracellular Ca²⁺ increases were unchanged in α-cells of αZnT8KO KO mice. ZnT8 is thus important in a subset of α-cells for normal responses to hypoglycemia and acts via Ca²⁺-independent mechanisms.     

Menadione-induced bleb formation in hepatocytes is associated with the oxidation of thiol groups in actin

Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) or the thiol oxidant, diamide (azodicarboxylic acid bis(dimethylamide)), resulted in the appearance of numerous plasma membrane protrusions (blebs) preceding cell death. Analysis of the Triton X-100-insoluble fraction (cytoskeleton) extracted from treated cells revealed a dose- and time-dependent increase in the amount of cytoskeletal protein and a concomitant loss of protein thiols. These changes were associated with the disappearance of actin and formation of large-molecular-weight aggregates, when the cytoskeletal proteins were analyzed by polyacrylamide gel electrophoresis under nonreducing conditions. However, if the cytoskeletal proteins were treated with the thiol reductants, dithiothreitol or beta-mercaptoethanol, no changes in the relative abundance of actin or formation of large-molecular-weight aggregates were detected in the cytoskeletal preparations from treated cells. Moreover, addition of dithiothreitol to menadione- or diamide-treated hepatocytes protected the cells from both the appearance of surface blebs and the occurrence of alterations in cytoskeletal protein composition. Our findings show that oxidative stress induced by the metabolism of menadione in isolated hepatocytes causes cytoskeletal abnormalities, of which protein thiol oxidation seems to be intimately related to the appearance of surface blebs.     

Ca(2+)-dependent and independent mitochondrial damage in hepatocellular injury

The alterations of mitochondrial membrane potential during the development of irreversible cell damage were investigated by measuring rhodamine-123 uptake and distribution in primary cultures as well as in suspensions of rat hepatocytes exposed to different toxic agents. Direct and indirect mechanisms of mitochondrial damage have been identified and a role for Ca2+ in the development of this type of injury by selected compounds was assessed by using extracellular as well as intracellular Ca2+ chelators. In addition, mitochondrial uncoupling by carbonylcyanide-m-chloro-phenylhydrazone (CCCP) resulted in a marked depletion of cellular ATP that was followed by an increase in cytosolic Ca2+ concentration, immediately preceding cell death. These results support the existence of a close relationship linking, in a sort of reverberating circuit, the occurrence of mitochondrial dysfunction and the alterations in cellular Ca2+ homeostasis during hepatocyte injury.     

Alterations in hepatocyte cytoskeleton caused by redox cycling and alkylating quinones

Quinones may induce toxicity by a number of mechanisms, including alkylation and oxidative stress following redox cycling. The metabolism of quinones by isolated rat hepatocytes is associated with cytoskeletal alterations, plasma membrane blebbing, and subsequent cytotoxicity. The different mechanisms underlying the effects of alkylating (p-benzoquinone), redox cycling (2,3-dimethoxy-1,4-naphthoquinone), and mixed redox cycling/alkylating (2-methyl-1,4-naphthoquinone) quinones on hepatocyte cytoskeleton have been investigated in detail in this study. Analysis of the cytoskeletal fraction extracted from quinone-treated cells revealed a concentration-dependent increase in the amount of cytoskeletal protein and a concomitant loss of protein thiols, irrespective of the quinone employed. In the case of redox cycling quinones, these alterations were associated with an oxidation-dependent actin crosslinking (sensitive to the thiol reductant dithiothreitol). In contrast, with alkylating quinones an oxidation-independent cytoskeletal protein crosslinking (insensitive to thiol reductants) was observed. In addition to these changes, a dose-dependent increase in the relative abundance of F-actin was detected as a consequence of the metabolism of oxidizing quinones in hepatocytes. Addition of dithiothreitol solubilized a considerable amount of polypeptides from the cytoskeletal fraction isolated from hepatocytes exposed to redox cycling but not alkylating quinones. Our findings indicate that the hepatocyte cytoskeleton is an important target for the toxic effects of different quinones. However, the mechanisms underlying cytoskeletal damage differ depending on whether the quinone acts primarily by oxidative stress or alkylation.     

The effects of terlipressin on regional hemodynamics and kidney function in experimental hyperdynamic sepsis

Background and Aims

Although terlipressin (TP) may improve renal function in cirrhotic patients, its use in sepsis remains controversial due to concerns about regional ischemia. We investigated the effects of TP on regional hemodynamics and kidney function in experimental hyperdynamic sepsis.

Methods

We studied thirteen merino ewes in a university physiology laboratory using a randomized controlled cross over design. We implanted flow probes around the pulmonary, circumflex coronary, superior mesenteric, renal and iliac arteries. We injected live Escherichia coli and induced hyperdynamic sepsis. We treated animals with either bolus vehicle or a single dose of TP (sTP = 1 mg). In a second group, after 1 mg of TP, two additional bolus injections (mTP) of 0.5 mg were given at 2 hourly intervals.

Main Results

sTP (1 mg) significantly increased mean arterial pressure (MAP) (74 to 89 mmHg; P<0.0001) creatinine clearance (31 to 85 mL/min; P<0.0001) and urine output (24 to 307 mL/hr) (P<0.0001). However, it decreased CO (5.7 to 3.9 L/min; p<0.0001), coronary blood flow (CBF) (43 to 32 mL/min; p<0.0001) and mesenteric blood flow (MBF) (944 to 625 mL/min; p = 0.004) and increased blood lactate (2.1 to 4.0 mmol/L; p<0.0001). Extra doses of TP caused little additional effect.

Conclusions

In hyperdynamic sepsis, bolus TP transiently improves MAP and renal function, but reduces CO, CBF and MBF, and increases blood lactate. Caution should be applied when prescribing bolus TP in septic patients at risk of coronary or mesenteric ischemia.     

Monte-Carlo Modeling of the Central Carbon Metabolism of Lactococcus lactis: Insights into Metabolic Regulation

Metabolic pathways are complex dynamic systems whose response to perturbations and environmental challenges are governed by multiple interdependencies between enzyme properties, reactions rates, and substrate levels. Understanding the dynamics arising from such a network can be greatly enhanced by the construction of a computational model that embodies the properties of the respective system. Such models aim to incorporate mechanistic details of cellular interactions to mimic the temporal behavior of the biochemical reaction system and usually require substantial knowledge of kinetic parameters to allow meaningful conclusions. Several approaches have been suggested to overcome the severe data requirements of kinetic modeling, including the use of approximative kinetics and Monte-Carlo sampling of reaction parameters. In this work, we employ a probabilistic approach to study the response of a complex metabolic system, the central metabolism of the lactic acid bacterium Lactococcus lactis, subject to perturbations and brief periods of starvation. Supplementing existing methodologies, we show that it is possible to acquire a detailed understanding of the control properties of a corresponding metabolic pathway model that is directly based on experimental observations. In particular, we delineate the role of enzymatic regulation to maintain metabolic stability and metabolic recovery after periods of starvation. It is shown that the feedforward activation of the pyruvate kinase by fructose-1,6-bisphosphate qualitatively alters the bifurcation structure of the corresponding pathway model, indicating a crucial role of enzymatic regulation to prevent metabolic collapse for low external concentrations of glucose. We argue that similar probabilistic methodologies will help our understanding of dynamic properties of small-, medium- and large-scale metabolic networks models.     

Menadione-induced cytotoxicity is associated with protein thiol oxidation and alteration in intracellular Ca2+ homeostasis

The toxicological implications of alterations in intracellular thiol homeostasis during menadione metabolism have been investigated using freshly isolated rat hepatocytes. A strict correlation between depletion of protein sulfhydryl groups and loss of cell viability was observed. Loss of protein thiols preceded cell death, and occurred more rapidly in cells with decreased levels of reduced glutathione. Depletion of protein thiols was also associated with inhibition of Ca²⁺ efflux from the cells and perturbation of intracellular Ca²⁺ homeostasis. It is proposed that the oxidative stress induced by menadione metabolism in isolated hepatocytes results in the depletion of both soluble and protein thiols, and that the latter effect is critically associated with a perturbation of Ca²⁺ homeostasis and loss of cell viability.     

Histological and immunohistochemical effects of Curcuma longa on activation of rat hepatic stellate cells after cadmium induced hepatotoxicity

The liver is a target for toxic chemicals such as cadmium (Cd). When the liver is damaged, hepatic stellate cells (HSC) are activated and transformed into myofibroblast-like cells, which are responsible for liver fibrosis. Curcuma longa has been reported to exert a hepato-protective effect under various pathological conditions. We investigated the effects of C. longa administration on HSC activation in response to Cd induced hepatotoxicity. Forty adult male albino rats were divided into: group 1 (control), group 2 (Cd treated), group 3 (C. longa treated) and group 4 (Cd and C. longa treated). After 6 weeks, liver specimens were prepared for light and electron microscopy examination of histological changes and immunohistochemical localization of alpha smooth muscle actin (αSMA) as a specific marker for activated HSC. Activated HSC with a positive αSMA immune reaction were not detected in groups 1 and 3. Large numbers of activated HSC with αSMA immune reactions were observed in group 2 in addition to Cd induced hepatotoxic changes including excess collagen deposition in thickened portal triads, interlobular septa with hepatic lobulation, inflammatory cell infiltration, a significant increase in Kupffer cells and degenerated hepatocytes. In group 4, we observed a significant decrease in HSC that expressed αSMA with amelioration of the hepatotoxic changes. C. longa administration decreased HSC activation and ameliorated hepatotoxic changes caused by Cd in adult rats.