Du Toit Loots

Experimental rodent models of type 2 diabetes: a review.

Due to the high prevalence of diabetes worldwide, extensive research is still being performed to develop new antidiabetic agents and determine their mechanisms of action. Consequently, a number of diabetic animal models have been developed and improved over the years, of which rodent models are the most thoroughly described. These rodent models can be classified into two broad categories: 1) genetically induced spontaneous diabetes models; and 2) experimentally induced nonspontaneous diabetes models. The popularity of using experimentally induced nonspontaneous models for diabetes research over that of the genetically induced spontaneous models is due to their comparatively lower cost, ease of diabetes induction, ease of maintenance and wider availability. The various experimentally induced type 2 diabetes (T2D) rodent models developed over the last 30-plus years for both routine pharmacological screening and mechanistic diabetes-linked research trials include: adult streptozotocin (STZ)/alloxan rat models, neonatal STZ/alloxan models, partial pancreatectomy models, long-term high-fat (HF) diet-fed models, HF diet-fed STZ models, nicotinamide/STZ models, intrauterine growth retardation (IUGR) models, the STZ-induced progressive diabetic model and monosodium glutamate (MSG)-induced model. The use of these models, however, is not without limitations. A T2D model should ideally portray an identical biochemical blood profile and pathogenesis to T2D in humans. Hence, this review will comparatively evaluate experimentally induced rodent T2D models considering the above-mentioned criteria, in order to guide diabetes research groups to more accurately select the most appropriate models given their specific research requirements.

Glycaemic control improves fibrin network characteristics in type 2 diabetes – A purified fibrinogen model

Diabetic subjects have been shown to have altered fibrin network structures. One proposed mechanism for this is non-enzymatic glycation of fibrinogen due to high blood glucose. We investigated whether glycaemic control would result in altered fibrin network structures due to decreased fibrinogen glycation. Twenty uncontrolled type 2 diabetic subjects were treated with insulin in order to achieve glycaemic control. Twenty age- and body mass index (BMI)-matched non-diabetic subjects were included as a reference group. Purified fibrinogen, isolated from plasma samples was used for analysis. There was a significant decrease in fibrinogen glycation (6.81 to 5.02 mol glucose/mol fibrinogen) with a corresponding decrease in rate of lateral aggregation (5.86 to 4.62) and increased permeability (2.45 to 2.85 x 10(-8) cm(2)) and lysis rate (3.08 to 3.27 microm/min) in the diabetic subjects after glycaemic control. These variables correlated with markers of glycaemic control. Fibrin clots of non-diabetic subjects had a significantly higher ratio of inelastic to elastic deformation than the diabetic subjects (0.10 vs. 0.09). Although there was no difference in median fiber diameter between diabetic and non-diabetic subjects, there was a small increase in the proportion of thicker fibers in the diabetic samples after glycaemic control. Results from SDS-PAGE indicated no detectable difference in factor XIIIa-crosslinking of fibrin clots between uncontrolled and controlled diabetic samples. Diabetic subjects may have altered fibrin network formation kinetics which contributes to decreased pore size and lysis rate of fibrin clots. Achievement of glycaemic control and decreased fibrinogen glycation level improves permeability and lysis rates in a purified fibrinogen model.

New sputum metabolite markers implicating adaptations of the host to Mycobacterium tuberculosis, and vice versa

In this study, a metabolomics research approach was used to identify new tuberculosis (TB) markers from sputum, in an attempt to better characterise the disease as well as the metabolic response of the host to Mycobacterium tuberculosis infection. After GCxGC-TOFMS analyses, various multivariate and univariate statistical methods were implemented to identify those compounds best describing the variation between the TB-positive and TB-negative patient groups. The interpretation of these new metabolite markers led to a number of new hypotheses, including: 1) support of the previously proposed citramalate cycle in M. tuberculosis; 2) the interaction of this cycle with an up-regulated glyoxylate cycle during pulmonary M. tuberculosis infection; 3) the increased utilisation of fatty acids and glutamate as alternative carbon sources by M. tuberculosis during pulmonary infection; 4) an alternative mechanism by which the host produces hydrogen peroxide via glucose oxidation, in order to eliminate the bacterial infection; 5) inhibition of the ETC due to pronounced oxidative stress during an active TB disease state, resulting in increased concentrations of various neurotransmitters and other metabolites previously associated with an inborn error of metabolism (MADD/GA type II); and 6) elevated concentrations of neurotransmitters associated with a number of previously described symptoms of TB.

Phytochemical contents and antioxidant capacities of two Aloe greatheadii var. davyana extracts.

Aloe greatheadii var. davyana (Asphodelaceae) is used among rural South African communities to treat arthritis, skin cancer, burns, eczema, psoriasis, digestive problems, high blood pressure and diabetes, despite very little supporting scientific evidence. Due to increased interest by both the scientific community and industry regarding the medicinal uses of this plant species, we identified, quantified and compared the phytochemical contents and antioxidant capacities of two extracts of A. greatheadii; a leaf gel extract (LGE) and a 95 % aqueous ethanol leaf gel extract (ELGE), using various modified extraction procedures, GC-MS and spectrophotometry. Apart from extensively characterizing this medicinal plant with regards to its organic acid, polyphenols/phenolic acid, alcohol, aldehyde, ketone, alkane, pyrimidine, indole, alkaloid, phytosterol, fatty acid and dicarboxylic acid contents and antioxidant capacities, we describe a modified extraction procedure for the purpose of general phytochemical characterization, and compare this to a 95 % aqueous ethanol extraction technique. From the results it is clear that A. greatheadii contains a variety of compounds with confirmed antioxidant capacity and other putative health benefits (such as blood glucose, cholesterol and cortisol lowering properties) relating to the prevention or treatment of diabetes, cardiovascular disease, cancer and hypertension. The results also indicate that separate ethyl acetate/diethyl ether and hexane extractions of the LGE, better serve for general phytochemical characterization purposes, and 95 % aqueous ethanol extraction for concentrating selective groups of health related compounds, hence justifying its use for biological in vivo efficacy studies.

A comparison of four sputum pre-extraction preparation methods for identifying and characterising Mycobacterium tuberculosis using GCxGC-TOFMS metabolomics.

In many pulmonary diseases, sputum is a valuable sample material for use in disease characterisation and diagnostics. However, due to its high viscosity and uneven consistency (lumpiness), it is difficult to obtain reproducible/repeatable results during compound extraction and analysis. We subsequently investigated and compared four sputum pre-extraction preparation methods using: 1) Sputolysin; 2) a combination of N-acetyl-l-cysteine and sodium hydroxide (NALC-NaOH); 3) NaOH alone, and 4) a simple ethanol homogenisation method, prior to sputum extraction and metabolomics analyses. The simple ethanol homogenisation approach proved to be the comparatively superior sputum pre-extraction preparation method, considering its repeatability, the number of characteristic compounds extracted, its ability to extract those compounds best differentiating the sample groups (Mycobacterium tuberculosis-spiked and clinically confirmed TB-positive patient samples from each of the controls respectively), and its detection limit. This developed methodology subsequently allows for accurate GC based analyses of sputum, and hence, could contribute significantly to the better characterisation or diagnostics of not only tuberculosis, but also potentially other pulmonary diseases, including, interstitial lung disease, cystic fibrosis, lung cancer, pneumonia and any other bacterial induced pulmonary diseases producing sputum.

The effect of glycaemic control on fibrin network structure of type 2 diabetic subjects

Diabetic subjects have been shown to have altered fibrin network structures. One possible cause may be fibrinogen glycation resulting in altered structure/function properties. We investigated the effect of glucose control on fibrinogen glycation and fibrin network structure in type 2 diabetes. Blood samples were taken from twenty uncontrolled diabetic subjects at baseline to determine the levels of fibrinogen glycation and fibrin network structures. The subjects were then treated with insulin until blood glucose control was achieved before end blood samples were taken. Twenty age- and BMI-matched non-diabetic subjects were included as a reference group. The diabetic subjects had significantly higher mean fibrinogen glycation at baseline than the non-diabetic subjects (7.84 vs. 3.89 mol glucose / mol fibrinogen; p < 0.001). This was significantly reduced during the intervention (7.84 to 5.24 mol glucose / mol fibrinogen; p < 0.0002) in the diabetic group. Both groups had high mean fibrinogen concentrations (4.25 and 4.02 g/l, diabetic and non-diabetic subjects respectively). There was no difference in fibrinogen concentration, porosity, compaction and kinetics of clot formation between the diabetic subjects and non-diabetic subjects at baseline, nor were there any changes during the intervention despite the reduced fibrinogen glycation. Fibrin network characteristics correlated well with fibrinogen but not with any markers of glycaemic control. Improved glycaemic control resulted in decreased fibrinogen glycation but not fibrinogen concentration. It seems as though porosity, compaction and kinetics of clot formation are more related to fibrinogen concentration than fibrinogen glycation in this model.

A metabolomics investigation of a hyper- and hypo-virulent phenotype of Beijing lineage M. tuberculosis

Despite a number investigations using rapid sequencing and comparative genomic techniques, attempting to characterise the phenomenon of varying degrees of virulence within the Mycobacterium tuberculosis species, the underlying causes for this still remain largely unexplained. The Beijing lineage of M. tuberculosis has received much attention due to a reported increased pathogenicity and global dissemination. In order to better understand these varying states of virulence, a GCxGC-TOFMS metabolomics research approach was used to compare the varying metabolomes of a hyper- and hypo-virulent Beijing strain of M. tuberculosis, and subsequently identify those metabolite markers differing between these strains. Multi- and univariate statistical analysis of the analysed metabolome data was used to identify those metabolites contributing most to the differences seen between the two sample groups. A general decrease in various carbohydrates, amino acids and lipids associated with cell wall structure and function, were detected in the hyper-virulent Beijing strain, comparatively. Additionally, components of mycothiol metabolism, virulence protein formation and energy production in mycobacteria, were also seen to differ when comparing the two groups. This metabolomics investigation is the first to identify the metabolite markers associated with an increased state of virulence, indicating increased metabolic activity, increased growth/replication rates, increased cell wall synthesis and an altered antioxidant mechanism, all of which would contribute to this organisms increased pathogenicity and survival ability.

Effects of dietary onion (Allium cepa L.) in a high-fat diet streptozotocin-induced diabetes rodent model.

Background/Aims: The present study was conducted to investigate the effects of two dietary doses of freeze-dried onion powder on diabetes-related symptoms in a high-fat (HF) diet streptozotocin (STZ)-induced diabetes rat model. Methods: Five-week-old male Sprague-Dawley rats were fed a HF diet for 2 weeks and then randomly divided into 4 groups as follows: HF control (HFC), diabetic control (DBC), onion low (ONL; 0.5%) and onion high (ONH; 2.0%). Diabetes was induced by an intraperitoneal injection of STZ (40 mg/kg body weight) in all groups except the HFC group. Results: After 4 weeks on the experimental diets, fasting blood glucose levels for both onion-fed groups were higher than in the DBC and HFC groups, albeit only significantly so (p < 0.05) in the ONL group. Serum insulin concentrations and insulin resistance were dose-dependently increased (however, not significantly so) in the onion-fed groups compared to the DBC group. Pancreatic β-cell function and liver glycogen concentrations were nonsignificantly higher in the DBC and ONH groups compared to the ONL group. Additionally, the ONH group had significantly higher lipid concentrations (except for high-density lipoprotein cholesterol) compared to all other groups. The ONL group showed a similar hyperlipidemic trend, however to a lesser extent, with only triglycerides significantly differing from those of the DBC and HFC groups. Conclusion: The results suggest that the HF onion diet may increase insulin secretion and consequently insulin resistance in a dose-dependent manner, resulting in a worsened hyperglycemic and hyperlipidemic diabetic state. We conclude that higher dietary fat may impair the antidiabetic effects of dietary onion intake as has been previously reported.

Self-reported alcohol intake is a better estimate of 5-year change in blood pressure than biochemical markers in low resource settings: the PURE study

Background: Despite criticism of self-reported alcohol intake, it is a valuable tool to screen for alcohol abuse as a risk factor for cardiovascular disease. We aimed to compare various self-reported estimates of alcohol use with &ggr;-glutamyltransferase (GGT) and percentage carbohydrate deficient transferrin (%CDT) considering their relationship with blood pressure changes (%BP) over a 5-year period in black South Africans. Method: We recruited 1994 participants and collected 5-year followed up data (N = 1246). Participants completed questionnaires on alcohol intake indicating their former and current alcohol use (‘yes’ response and ‘no’ if alcohol was never used). We assessed alcohol intake (in g) using a quantified food frequency questionnaire. We collected blood samples and measured GGT and %CDT. Brachial BP (bBP) was measured at baseline and follow-up and central BP (cBP) at follow-up only. Results: Self-reported alcohol intake was significantly associated with the 5-year change in bBP before and after adjusting for confounders (%bSBP: R2 = 0.263, &bgr; = 0.06, P = 0.023; %bDBP: R2 = 0.326, &bgr; = 0.08 P = 0.005), as well as cSBP (R2 = 0.286, &bgr; = 0.09, P = 0.010) and central pulse pressure (R2 = 0.254, &bgr; = 0.06, P = 0.020). GGT and %CDT correlated well with self-reported alcohol intake (r = 0.44; P = 0.001; r = 0.34 P = 0.001), but did not associate significantly with %bBP or cBP at follow-up. Conclusion: Self-reported alcohol use was strongly associated with a 5-year increase in BP in Africans with a low socio-economic status. This was not found for biochemical measures, GGT and %CDT. Self-reported alcohol intake could be an important measure to implement in primary healthcare settings in middle to low income countries, where honest reporting is expected.

Melatonin prevents the free radical and MADD metabolic profiles induced by antituberculosis drugs in an animal model

Abstract:  The objective was to determine the effect of combined antituberculosis (anti‐TB) drug therapy and an antioxidant, melatonin, on the free radical and organic acid profiles in an experimental rat model. A combined anti‐TB drug, Rifater, consisting of 12.0 mg rifampicin, 0.8 mg isoniazid, and 23.0 mg pyrazinamide and 18.56 μg melatonin/kg body weight per day (corresponding to average physiological human intake) were orally administered to Sprague–Dawley rats. Hydroxyl radical production was monitored by quantifying 2,3‐dihydroxybenzoic acid produced after intraperitonial sodium salicylate injections. Organic acid extractions and gas chromatography‐coupled mass spectrometry analyses were performed on collected urine samples. The results show hydroxyl radicals (P = 0.0019) and organic acids (P‐value range: 0.037 to <0.001), characteristic of a multiple acyl‐CoA dehydrogenase defect (MADD), were elevated with Rifater treatment and these elevations were significantly lowered with melatonin pretreatment (P‐value range: 0.031 to <0.001), probably because of its inherent antioxidant activity. We conclude that hydroxyl radical production and an increased organic acid profile induced by anti‐TB medication indicates inhibition of the electron transport chain. We also conclude that free radicals leading to clinical symptoms associated with an MADD metabolic profile induced by anti‐TB treatment could be alleviated by melatonin intervention.