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- what is the experimental design used in this article? Is this an appropriate experimental design and why?

- what statistical tests were used and were they appropriate and why?


Multiple experiments in male Wistar rats were designed to clarify the role of mitochondrial dysfunction in the mechanisms of oxidative stress-related diseases and toxicity-induced pathologies. In this particular report, 21 male Wistar rats were supplemented ad libitum with either As3+ or Cr3+ salts in drinking water to assess insulin secretion patterns in vivo and in vitro, mitochondrial dysfunction, oxidative stress, liver damage, basal insulin and glucose tolerance curves, among other parameters. Results were compared with a control group without any metal supplementation. The CrCl3 supplements were more invasive of metabolism and had a stronger effect on mitochondrial dysfunction than As3+, despite that both seem to use similar mechanisms of toxicity; viz.: binding to thiol or -SS- group in enzymes and proteins, and releasing oxidant species during their redox-cycling and metabolic activation processes, e.g., by cytochrome p450 in liver. Results support our aim to prove the influence of oxidative stress-induced mitochondrial dysfunction on glycemic control.

Nutrition: 1997; 13:965-970

This study is part of broader studies designed to clarify mechanisms of pathologies mediated or associated with oxidative stress, particularly those of diabetogenesis and the responses to an innovative diet therapy for non-insulin-dependent diabetes mellitus (NIDDM). In order to develop our hypothesis of oxidative damage-induced mitochondrial dysfunction, we designed multiple experiments in rats. We report results related to the effects of supplementation with Cr3+ and As3+ ions at increasing concentrations gradually reaching the toxic threshold. These results provide important clues that we used to design the aforementioned therapy and are presented here as partial findings. These will be included in our forthcoming proposed clinical trial for NIDDM-therapy and for treatment/prevention of other oxidative stress-induced mitochondrial dysfunctions, which may underlie the etiology of diseases such as Alzheimer's or those associated with deletions in mitochondrial DNA and Kearns-Sayre syndrome and Pearson's syndrome.

Although our methods focus on how changes in mitochondrial respiration contribute to metabolic alterations, the goals of this study were 1) to determine insulin secretion patterns in rats subjected to chronic, ad libitum supplementation with As3+ or with Cr3+, both in vivo and in vitro (i.e., released from isolated Langerhans islets); 2) to obtain further evidence on the mechanisms by which As-toxicity (which also includes its insulin-binding interference) and Cr-mediated insulin sensitivity might affect insulin release and efficiency (previously we had compared the difference between therapeutic forms of organic-Cr (brewer's yeast) and damaging forms of ionic Cr3+ as well as the synergic toxicity of combined supplementation with both As3+ and Cr3+); 3) to split and compare insulin-release differences in vivo and in vitro, under the effects of such supplementation and to analyze the difference between in vivo short- and long-term responses; 4) to demonstrate the mitochondrial respiration modifications induced by metal ion supplementation in the long-term in vivo responses to glucose challenge and in carbohydrate metabolic pathways that may play key roles in diabetogenesis and in diabetic complications; and 5) to differentiate toxicity and homeostatic dysregulation resulting from uncoupling at energy conservation sites or interferences at coupling factors, from those due to ion-binding to enzymes and/or metabolic intermediates as happens in arsenolysis. Mitochondrial activity plays a key role in metabolic regulation and in the onset of most oxidative stress-mediated dysregulation, including aging and genetically and environmentally induced pathologies of degenerative diseases; therefore we assessed it through measurement of respiratory parameters (state 3, state 4, their respiratory control ratio (RCR) quotient, and adenosine diphosphate/oxygen [ADP/O] ration).

Materials and Methods:
In Vivo Models:
21 male Wistar rats were divided into 3 groups of 7: the control group being fed a standard diet and the other 2 groups fed either Cr3+ or As3+ in drinking water, at concentrations increasing from (in the first week) 17.75 mg/L As2O3 or 177.5 mg/L CrCl3, to (at the end of the 8th week) 100 mg/L As2O3 or 1000 mg/L CrCl3.

Following a glucose load, the glucose tolerance curve was determined to assess the type of glycemic behavior induced by As3+ or Cr3+ supplementation. Serum glucose variations over time were determined as area under curve (AUC) for a period of 3 hours following the glucose challenge.

In Vitro Insulin Release:
Rat pancreas islets were isolated and their release of insulin measured under varying conditions.

Isolation of Mitochondria:
Mitochondria were obtained from the livers of sacrificed rats. An average of 100 mg of mitochondrial protein was obtained from 100 mg of wet liver.

Mitochondrial Respiratory Ratio:
Oxygen consumption was measured in suspensions of mitochondria. Oxygen consumption with endogenous substrate, representing state 1 of mitochondrial respiration, was recorded for 1 minute. State 2 is reached upon ADP addition and is characterized by slow respiration rate and the substrate level is the rate-limiting factor. State 3 of mitochondrial respiration is the fastest rate and is induced by addition of additional substrate so that the respiratory chain efficiency becomes the rate-limiting factor. State 4, with a lower oxygen consumption rate, occurs after ADP exhaustion. Oxygen exhaustion characterizes state 5 of respiration breakdown, with oxygen as the rate-limiting factor. The RCR (ratio of oxygen consumption in the presence and absence of ADP when substrate is not a limiting factor) is calculated as the ratio of the rates of oxygen consumption during states 3 and 4 (= rate 3/rate 4).

Statistical Analyses:
One-way analyses of variance using Scheffe's method for multiple comparisons and alpha of 0.05 were employed.

Glucose Clearance:
Compared to control rats, rats fed supplemental As3+ exhibited delayed glucose clearance but much lower maximum plasma glucose concentrations. Supplemental chromium had no effect on plasma glucose concentrations following a glucose load.

Table 1:
Fasting plasma insulin concentrations were (mean +/- SD):
control 20 +/- 5.5 (A)
+As3+ 21.5 +/- 6.0 (A)
+Cr3+ 7.5 +/- 0.5 (B)
(means with different letters are different, p<0.05)

Cr3+ supplementation significantly lowered insulin release in response to glucose challenge.

Table 2:
In vitro release of insulin by isolated pancreatic islets in response to exposure to glucose (mean +/- SD):
2.8 mM glucose: 16.7 mM glucose:
control 1400 +/- 25 (A) 1500 +/- 50 (A)
+As3+ 1700 +/- 100 (B) 1700 +/- 30 (B)
+Cr3+ 1375 +/- 50 (A) 1450 +/- 60 (A)
(means with different letters are different, p<0.05)

In vitro insulin release from isolated pancreatic islets was glucose-dependent and varied with treatment. Insulin release was increased above control in the presence of Ar3+ and was decreased in the presence of Cr3+. Insulin release may have failed to increase with increasing glucose concentration in As3+-supplemented islets because of exhaustion of islet-stocks of proinsulin. Insulin release may have failed to increase with increasing glucose concentration in ACR3+-supplemented islets because of stabilization of receptor binding.

Table 3:
Mitochondrial oxygen consumption rates in state 3 (mean +/- SD):

glutamate + malate succinate
control 85 +/- 45 (A) 120 +/- 55 (C)
+As3+ 80 +/- 5 (A) 95 +/- 15 (A)
+Cr3+ 40 +/- 10 (B) 50 +/- 7 (B)
(means with different letters are different, p<0.05)

Both As3+ and Cr3+ inhibited state 3 of mitochondrial respiration, with a stronger effect by chromium.

Table 4:
Mitochondrial oxygen consumption rates in state 4 (mean +/- SD):

glutamate + malate succinate
control 18 +/- 9 (A) 25 +/- 3 (B)
+As3+ 17 +/- 3 (A) 24 +/- 6 (B)
+Cr3+ 13 +/- 4 (A) 18 +/- 3 (C)
(means with different letters are different, p<0.05)

Both As3+ and Cr3+ inhibited state 4 of mitochondrial respiration, with a stronger effect by chromium.

Table 5:
Mitochondrial respiratory control ratios (mean +/- SD):

glutamate + malate succinate
control 8 +/- 2.0 (A) 5.8 +/- 1.0 (B)
+As3+ 5.5 +/- 0.6 (B) 4 +/- 0.8 (C)
+Cr3+ 3.8 +/- 0.2 (C) 3.6 +/- 0.6 (C)
(means with different letters are different, p<0.05)

Respiratory control ratio was significantly decreased in the presence of either As3+ or Cr3+, with the greatest effect accompanying exposure to Cr3+.

Table 6:
Mitochondrial ADP/O consumption ratio (mean +/- SD):

glutamate + malate succinate
control 3 +/- 0.8 (A) 1.7 +/- 0.4 (C)
+As3+ 2.4 +/- 0.1 (B) 2.0 +/- 0.2 (C)
+Cr3+ 2.4 +/- 0.2 (B) 1.6 +/- 0.3 (C)
(means with different letters are different, p<0.05)

ADP/O consumption ratio (an index of the extent of coupling of oxidation and phosphorylation) was decreased in the presence of either As3+ or Cr3+, indicating that both of these ions inhibited mitochondrial respiration.

One reason for As3+-induced glycemia and dampening of glucose clearance from plasma might simply be the As-mediated inhibition of pyruvate dehydrogenase catalysis to form Acetyl-CoA as the entering point of glucose into the Krebs cycle, with concomitant production of NADH, which is also necessary for converting pyruvate into lactic acid in anaerobic glycolysis. Thus, excess pyruvate diverts back to glucose-6-phosphate, which converts into glucose upon hydrolysis and is released from hepatocytes and other tissues back into the bloodstream. The opposite effect is necessary for glycemic control and needs to be achieved to maintain a high NADH/NAD ratio in therapy. The binding of our trivalent cations to thiol groups lowers NADH/NAD ratio by several pathways: by lowering GSSG-reductase activity, thus depleting GSH; by inhibiting thiolase activity, leading to NASH depletion; and by lowering Acetyl-CoA through pyruvate dehydrogenase and thiolase inhibition, leading to a lower NADPH production in the Krebs cycle. Implications of this depletion in hyperglycemia, insulinopenia, and diabetes treatment have been suggested and will be discussed elsewhere.

Chromium effects seem to delay the insulin action and extend its receptor binding, supporting the theory of a ternary complex with membrane thiol groups and interchain disulfide groups from the A-chain of insulin, which does not exclude several forms of bioactive Cr and non-Cr molecular complexes in insulin enhancement.

The toxic effects on mitochondrial respiration are very relevant to understanding the influence of mitochondrial dysfunctions on oxidative stress and their effects on overall metabolic regulation mechanisms. For example, let us consider that a lowering in respiratory control diverts Acetyl-CoA into increased citrate export to cytosol and fatty acid synthesis. However, a simultaneous uncoupling of ATP production introduces multiple dysregulations in ATP-mediated transport and catalysis. This may lead to accumulation of free fatty acids in serum as seen in insulin-dependent diabetes mellitus.

Both in vivo and in vitro experiments were used throughout; in vivo there is a release of hepatic growth factor to repair liver damage, which does not occur in vitro. Oxidative stress arising from chronic illness such as diabetes, or from impaired mitochondrial activity, is mimicked by our trivalent-cation supplementation both at the level of releasing reactive oxygen species and free radicals into the mitochondrial matrix and for nuclear damage: e.g., the metabolic activation of As3+ to AsO4 explains why arsenate, which binds DNA-activating oncogenes, is more genotoxic and carcinogenic than Cr3+, which binds DNA-inducing mutations, so the latter is more mutagenic and more invasive of metabolic pathways through its binding to disulfide bridges in thiol proteins/enzymes.

The mitochondrial RCR is more inhibited by Cr3+ than by As3+ as shown in Table 5, and it is due to a stronger inhibition of state 3 rate than of state 4 (Tables 3 and 4). Metabolic activation of As3+ to AsO4 followed by arsenate replacement for phosphate in all phosphorolytic reactions leads to arsenolysis, which affects processes such as oxidative phosphorylation in this way: instead of 1,3-diphosphoglycerate (which transfers 1-phosphoryl to ADP yielding ATP), the As-polluted mitochondria form 1-arseno-3-phosphoglycerate, which allows oxidation of 3-phospho-glyceraldehyde but uncouples ATP synthesis at the 1-phosphoryl position. Furthermore, the transient formation of arsenate esters allows enzymes that normally act on phosphorylated substrates to catalyze a slow reaction of the unphosphorylated substrate, which would not occur in the absence of AsO4. Other species created by metabolic activation, e.g., arsenite, react quickly with dithiols such as lipoic acid, thus blocking enzymes requiring it. This mechanism explains respiratory chain inhibition by blocking mitochondrial dehydrogenases and is common to Cr3+ and As3+, and our findings further confirm the following:

1. state 3 is more inhibited than state 4;
2. glutamate + malate are more inhibited than succinate;
3. both ions act on sites 1 and 2 of ATP synthesis;
4. Cr3+ is a more potent inhibitor than As3+, probably by means of stronger disulfide binding and a synergic oxidative stress induction both at the level of NADPH depletion and GSSG stabilization, thus depleting GSH by interfering with the process:

NADPH + H+ (by action of GSSG-reductase) to NADP+ + 2GSH

5. thus, the lowering of respiratory control is also stronger for Cr3+, as shown in Table 5;
6. ADP/O ratios represent the number of ATP moles generated by each mole of O used in the respiration, theoretically 3 for glutamate + malate (one at each energy-conservation site) and 2 for succinate, which joins the pathway after site 1. After careful electrode calibrations we found for As3+, values of 2.35 for glutamate + malate and 2 for succinate, confirming that inhibition is at site 1 by NADH depletion. Similar values found for Cr3+ show that the difference seems less due to mechanism, but more to synergic oxidative damage;
7. Table 1 shows no significant increase of basal insulin in As-supplemented rats, probably because of a homeostatic adaptation to impairment of glucose use in the Krebs cycle, attributed to A3+ depletion of Acetyl-CoA synthesis by pyruvate dehydrogenase. In vitro, As3+ induces sharp insulin release from islets in such a way that an increase in glucose concentration cannot significantly enhance it further (Table 2). However, Cr3+-induced glycemia chronically associated glucose clearance by sorbitol pathways depletes (NADPH/NADP+), while raising (NADH/NAD+), a situation that seems atherogenic and may be responsible for diabetic angiopathies and complications, as pointed out by some investigators.

We emphasize that all care must be taken when experiments in animals are used to draw conclusions that are valid for humans. In this case we only assess the oxidative stress induced by enhanced oxygen consumption in mitochondria where ATP synthesis is uncoupled and antioxidants in the electron chain complexes are depleted. Under these circumstances, based on our own and other colleagues' results, we assume that the overproduction of reactive oxygen species and the untrapped free radicals induce damage in mitochondrial DNA, initiating a loop of oxidative stress that reaches nuclear DNA, whose damage affects the expression of proteins and DNA-repair enzymes. Some of these affected proteins are responsible for the antioxidant defense system and for nuclear repair, and belong to the mitochondrial respiratory chain itself, thus amplifying the loop of oxidative damage that enters a vicious circle of oxidative damage responsible not only for diabetic complications, but also for many symptoms in degenerative diseases associated with oxidative stress.

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what is the experimental design used in this article? Is this an appropriate experimental design and why?
<br><br>The main aim was to lend support to the hypothesis that oxidative stress-induced mitochondrial dysfunction influences glycemic
<br><br>control. To prove the presence of mitochondrial dysfunction, they
<br><br>treated rats with Cr+3 and As+3 ions at increasing conc. and
<br><br>compared it to a control group.These ions mimic the effects of
<br><br>oxidative stress conditions.Studies we done in both in-vivo and in-vitro conditions.
<br><br>They compared the insulin ...

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