Scott Heffernan

Changes in Hepatic Glutathione Metabolism in Diabetes

Glutathione is important in the regulation of the redox state, and a decline in its tissue level has often been considered to be indicative of increased oxidative stress in diabetes. In this study of diabetic rats, the level of hepatic glutathione was normal unless food intake was restricted. Thus, the previous report of a reduction in hepatic glutathione in diabetes is likely to be the result of food deprivation rather than diabetes alone. In contrast to changes characteristic of oxidative stress, the efflux of glutathione in bile from diabetic animals was significantly decreased, whereas hepatic mixed disulfides were unchanged, and the hepatic γ-glutamyltransferase activity was considerably increased. These changes were not reproduced by food deprivation. The decrease in biliary excretion of glutathione in diabetes may reflect an attempt to conserve glutathione by activation of the hepatic γ-glutamyl cycle. We conclude that the disturbances of glutathione metabolism in diabetes are not typical of those seen in oxidative stress or food restriction.

Deficiency of Ascorbic Acid in Experimental Diabetes: Relationship With Collagen and Polyol Pathway Abnormalities

The plasma and tissue concentration of ascorbic acid (AA) is reduced in diabetes. This study was designed to investigate the mechanism and significance of this phenomenon. The low plasma AA concentration of diabetic rats can be normalized by dietary AA supplement (20–40 mg/day), a dosage approximately equal to the maximal synthetic rate of this substance in the rats. Treatment of diabetic rats with this regime prevented the decrease in activity of granulation tissue prolyl hydroxylase (PRLase), an AA-dependent enzyme required for maintaining the normal properties of collagen. The decreased plasma AA concentration and granulation tissue PRLase activity in diabetes can also be normalized by the aldose reductase inhibitor tolrestat. We conclude that in diabetic animals there is a true deficiency of AA that may be responsible for some of the changes of collagen observed in diabetes. Treatment with AA or an aldose reductase inhibitor may prevent some of the diabetic complications with underlying collagen abnormalities.

Ascorbic Acid Metabolism and Polyol Pathway in Diabetes

It has been reported previously that the plasma concentration of ascorbic acid (AA) is reducedin streptozocin-induced diabetic rats and can be normalized by treatment with the aldose reductase inhibitor tolrestat. This study was designed to investigate further the relationship between the polyol pathway and AA metabolism in diabetic rats. Disturbance of AA metabolism was demonstrable after 1 wk of diabetes. Dietary myo-inositol supplementation was effective in normalizing plasma AA levels, as was treatment with tolrestat. In untreated diabetes, despite low plasma AA concentration, there was increased urinary excretion of AA that was reversed by treatment with either tolrestat or myoinositol. In contrast, AA supplementation normalized plasma AA concentrations while further increasing urinary AA excretion. The abnormality of AA metabolism was less severe in galactose-fed rats, which had normal plasma AA levels and only minor increases in urinary AA excretion. These studies demonstrated a disturbance in the regulation of plasma and urinary AA concentration in experimental diabetes and confirmed the relationship of AA with the polyol pathway. Because AA has many important biological functions, abnormalities of AA metabolism could be important in the pathogenesis of some diabetic complications. The interaction of the polyol and AA pathways suggests that this could be another site of action for aldose reductase inhibitors.

Renal connective tissue growth factor correlates with glomerular basement membrane thickness and prospective albuminuria in a non-human primate model of diabetes: possible predictive marker for incipient diabetic nephropathy

UNLABELLED Diabetic renal disease is characterized by accumulation of extracellular matrix, glomerulosclerosis, and tubulointerstitial fibrosis. Connective tissue growth factor (CTGF) is implicated in these changes, as it contributes to new matrix synthesis and is increased in the diabetic kidney. CTGF also inhibits mesangial matrix degradation through up-regulation of the tissue inhibitor of matrix metalloproteinase 1 (TIMP-1). In a non-human primate model of diabetes, we determined whether the level of renal CTGF protein before development of albuminuria correlated with renal matrix and TIMP-1 changes and whether renal CTGF predicts progression to albuminuria. METHODS In a group of diabetic (n=9) and control (n=6) baboons after a 5-year duration of diabetes, renal tissue CTGF and TIMP-1 were detected by immunohistochemistry and compared with glomerular basement membrane (GBM) thickness and mesangial volume measurements from electron photomicrographs of renal biopsies. Urinary albumin levels were measured at 5 and 10 years of diabetes. RESULTS GBM thickness, CTGF protein, and TIMP-1 protein were increased after 5 years of diabetes (each P<.05). Tubular fibronectin scores correlated with tubular CTGF scores (r=0.72, P=.002). In diabetic animals, GBM thickness correlated with tubular and total CTGF levels (P=.002 and P=.04, respectively), whereas mesangial cell and total matrix volume correlated with glomerular TIMP-1 (P=.02 and P=.01, respectively). Tubular CTGF scores (P=.008) and GBM thickness (P=.03) at 5 years in diabetes each predicted the degree of albuminuria at 10 years. CONCLUSIONS These results suggest that early increases in renal CTGF protein contribute to incipient diabetic nephropathy and that renal CTGF may have utility as an early marker for progression to dysfunction in the diabetic kidney.

Tumor necrosis factor α induces a model of preeclampsia in pregnant baboons (Papio hamadryas).

Preeclampsia is a common disease of pregnancy characterised by maternal hypertension and proteinuria. Abnormal placentation in early pregnancy and abnormal cytokine and anti-angiogenic factor expression are thought to contribute to the clinical syndrome of endothelial dysfunction evident in the second half of gestation. The mechanisms underlying both the placental pathology and its translation to the maternal clinical syndrome are not fully understood. A model of preeclampsia manifest by clinically evident endothelial dysfunction (increased blood pressure and proteinuria) was induced by administration of low-dose TNF-α for 2weeks at mid-gestation in pregnant baboons (Papio hamadryas). Blood pressure was monitored continuously and remotely by intra-arterial radiotelemetry. Following TNF-α infusion, there was an increase in systolic and diastolic blood pressure and development of proteinuria in pregnant treated animals, but not in pregnant saline controls nor in non-pregnant TNF-α treated animals. The treated pregnant animals also developed elevated plasma soluble FMS-like tyrosine kinase-1 (sFLT-1) and increased placental mRNA expression of sFLT-1 and soluble endoglin (sEng). These results clearly demonstrate that the cytokine TNF-α can induce the clinical and biochemical features of human preeclampsia. The results identify a link between cytokines, placental dysfunction and endothelial dysfunction resulting in a loss of maternal blood pressure control.

Interaction of Ascorbic Acid and Glucose on Production of Collagen and Proteoglycan by Fibroblasts

Collagen and proteoglycans are two major constituents of the extracellular matrix, and their abnormalities have been incriminated in the pathogenesis of diabetic complications. A decrease of plasma ascorbic acid has been reported in diabetes and thus may play a role in the collagen and proteoglycan abnormalities in diabetes. Ascorbic acid and glucose share structural similarity, and their metabolism may interact at the level of membrane transport and cellular action. In this study, we used a fibroblast culture system to explore this possibility. Ascorbic acid increased collagen and proteoglycan both in the culture medium and the cell layer. This stimulatory action of ascorbic acid was inhibited by the presence of glucose at a concentration of 25 mM. The effect of high glucose concentration was not mediated by inhibition of ascorbic acid uptake by fibroblasts. Insulin is able to abolish this inhibitory action of glucose on collagen production, but the precise mechanism is unclear. These results show that the high glucose concentration in diabetes can impair the action of ascorbic acid at the cellular level. This may further accentuate the problem of decreased availability of this vitamin as a result of its low plasma concentration.

Abnormalities of ascorbic acid metabolism and diabetic control: differences between diabetic patients and diabetic rats.

Ascorbic acid is required in the synthesis of collagen and is also an important anti-oxidant. In a previous study, plasma ascorbic acid concentration was found to be decreased in diabetic patients but there was no relationship with blood glucose level. In the current study of diabetic patients, both plasma ascorbic acid and its urinary excretion correlated inversely with glycosylated hemoglobin level. Plasma ascorbic acid was also lower in diabetic rats but urinary ascorbic acid was elevated. The divergent trend in urinary ascorbic acid excretion observed in diabetic patients and diabetic rats may be due to difference in the ability of these two species to synthesize ascorbic acid. Difference in renal reabsorption of ascorbic acid may also be a relevant factor. The lower plasma and urinary ascorbic acid levels in diabetic patients with more severe hyperglycaemia indicates that this group of patients is particularly at risk of developing deficiency of this vitamin. As ascorbic acid has many important functions in the body, it may be necessary to supplement this vitamin in patients with chronically poorly controlled diabetes.

Functional and structural abnormalities in the nerves of Type I diabetic baboons: aminoguanidine treatment does not improve nerve function

Aims/hypothesis. To improve understanding of the pathophysiology of diabetic neuropathy and to establish a primate model for experimental studies, we examined nerve changes in baboons with Type I (insulin-dependent) diabetes mellitus. We also examined the effect of aminoguanidine (an inhibitor of the formation of advanced glycation end products) on nerve function.¶Methods. Male baboons (Papio hamadryas) were assigned to four groups; control, diabetic, control and diabetic treated with aminoguanidine. Diabetes was induced with streptozotocin (60 mg/kg, intravenous). Insulin and aminoguanidine (10 mg/kg) were injected subcutaneously daily. Motor and sensory nerve conduction velocity was measured using standard techniques. Autonomic function was examined by measuring heart rate response to positional change. Sural nerve morphometry was analysed in the diabetic group (mean duration 5.5 years) along with their age-matched controls.¶Results. The diabetic groups were smaller in size with a mean HbA1 c of 8.9 ± 1.2 %. The nerve conduction velocity and heart rate response was reduced in the diabetic groups. Morphometric analysis of the diabetic sural nerve showed smaller axon diameter (2.99 ± 0.06 μm vs 3.29 ± 0.06 μm; p < 0.01) accompanied by thinner myelin (1.02 ± 0.02 μm vs 1.15 ± 0.02 μm, p < 0.01) with no change in the axon density. Treatment with aminoguanidine for 3 years had no effect on glycaemic control and did not restore conduction velocity or autonomic dysfunction in the diabetic animals, contrary to the studies in rats.¶Conclusions/interpretation. These results show that the primate is a good model to study diabetic neuropathy and suggest that the accumulation of advanced glycation end products are not an early mechanism of nerve damage in this disorder. [Diabetologia (2000) 43: 110–116]

Placental Growth Factor Reduces Blood Pressure in a Uteroplacental Ischemia Model of Preeclampsia in Nonhuman Primates

An imbalance in the angiogenesis axis during pregnancy manifests as clinical preeclampsia because of endothelial dysfunction. Circulating soluble fms-like tyrosine kinase 1 (sFLT-1) increases and placental growth factor (PlGF) reduces before and during disease. We investigated the clinical and biochemical effects of replenishing the reduced circulating PlGF with recombinant human PlGF (rhPlGF) and thus restoring the angiogenic balance. Hypertensive proteinuria was induced in a nonhuman primate (Papio hamadryas) by uterine artery ligation at 136 days gestation (of a 182-day pregnancy). Two weeks after uteroplacental ischemia, rhPlGF (rhPlGF, n=3) or normal saline (control, n=4) was administered by subcutaneous injection (100 &mgr;g/kg per day) for 5 days. Blood pressure was monitored by intra-arterial radiotelemetry and sFLT-1 and PlGF by ELISA. Uteroplacental ischemia resulted in experimental preeclampsia evidenced by increased blood pressure, proteinuria, and endotheliosis on renal biopsy and elevated sFLT-1. PlGF significantly reduced after uteroplacental ischemia. rhPlGF reduced systolic blood pressure in the treated group (−5.2±0.8 mm Hg; from 132.6±6.6 mm Hg to 124.1±7.6 mm Hg) compared with an increase in systolic blood pressure in controls (6.5±3 mm Hg; from 131.3±1.5 mm Hg to 138.6±1.5 mm Hg). Proteinuria reduced in the treated group (−72.7±55.7 mg/mmol) but increased in the control group. Circulating levels of total sFLT-1 were not affected by the administration of PlGF; however, a reduction in placental sFLT-1 mRNA expression was demonstrated. There was no significant difference between the weights or lengths of the neonates in the rhPlGF or control group; however, this study was not designed to assess fetal safety or outcomes. Increasing circulating PlGF by the administration of rhPlGF improves clinical parameters in a primate animal model of experimental preeclampsia.

The effects of aminoguanidine on renal changes in a baboon model of Type 1 diabetes

BACKGROUND The efficacy of aminoguanidine (AG) on primary prevention of diabetic nephropathy was investigated in a nonhuman primate model of Type 1 diabetes over a period of 4 years. METHODS Adolescent male baboons (Papio hamadryas) were assigned to four groups: control, diabetic, and control and diabetic treated with AG. Diabetes was induced with streptozocin (60 mg/kg) and treated with insulin to maintain a mean HbA1c level of about 9%. AG was given subcutaneously (10 mg/kg) each day. All animals had annual renal biopsies and 24-h urine collections for measurements of glomerular basement membrane (GBM) thickness, fractional mesangium volume (FMV), albumin excretion rate (AER), and creatinine clearance. Glomerular filtration rate (GFR) and renal plasma flow (RPF) were also determined. RESULT The diabetic animals had increased GBM after 2 years of diabetes, but there was no increase in FMV over the study period. AG prevented the thickening of GBM at the 3- and 4-year time points. AG and diabetes synergistically increased the GFR. All diabetic animals developed increased albuminuria during the study although lower than the conventionally accepted microalbuminuria range. AG was not able to prevent this and, in fact, led to the nondiabetic animals also developing albuminuria. CONCLUSION This is the first study to investigate the early use of AG in ameliorating renal damage in a primate model of Type 1 diabetes. The structural and functional changes in the kidney of these animals resemble those seen in the early stages of the human disease. AG was able to significantly reduce the thickening of GBM due to diabetes. This may suggest a potential role for this in primary prevention of diabetic nephropathy in the future.