Glucokinase gene transfer to skeletal muscle of diabetic Zucker fatty rats improves insulin-sensitive glucose uptake.

Skeletal muscle has a prime role in glucose homeostasis. We have previously demonstrated that adenovirus-mediated glucokinase (GK) gene transfer to skeletal muscle of Wistar rats enhances muscle glucose uptake and whole body glucose disposal under conditions of hyperglycemia and hyperinsulinemia. In this study, we have tested whether GK gene transfer to the muscle of the Zucker Diabetic Fatty rat (ZDF), a genetic model of obesity and type 2 diabetes, could improve glycemic control and prevent the onset of hyperglycemia in obese males. We show that GK delivery results in a doubling of total gastrocnemius muscle glucose phosphorylating activity 9 weeks after gene transfer. GK-treated rats exhibited slightly reduced weight and normal insulin-sensitive glucose uptake, as assessed during an insulin tolerance test, whereas age-matched rats treated with a control virus were clearly insulin resistant. The improved glucose uptake in GK-expressing rats was associated with higher gastrocnemius lactate content, whereas glycogen and triacylglyceride (TAG) levels were unmodified. Remarkably, GK-treated rats showed increased expression of both hexokinase II (HKII) and GLUT4, in accordance with a glucose-dependent regulation of these proteins. Thus, our data show that delivery of GK, despite improving insulin-sensitive glucose disposal in muscle, is not sufficient to prevent or delay the appearance of elevated glucose and insulin levels associated with severe obesity in male ZDF animals.

Neuronal nitric oxide synthase activity in the paraventricular nucleus buffers central endothelin-1- induced pressor response and vasopressin secretion.

Endothelin 1 (ET-1) injected into the lateral cerebral ventricle increases sympathetic output, arterial pressure and plasma vasopressin (AVP). These responses are mediated by glutamatergic inputs and inhibited by gamma-amino-butyric acidergic inputs in the paraventricular nucleus (PVN). It has been suggested that nitric oxide enhances these gamma-amino-butyric acidergic inhibitory inputs. The present studies were designed to test the hypothesis that decreasing neuronal nitric oxide synthase (nNOS) activity within the PVN will potentiate ET-1-induced increases in arterial pressure and alter plasma AVP secretion. Male Long Evans rats underwent adenoviral gene transfer of beta-galactosidase, Ad.CMV.beta-gal (6.25 x 10(4) pfu/PVN; control, n = 5) or injection with DNA plasmids encoding dominant-negative forms of nNOS (RSV hemedomain or RSV heme-RedF; mutant, n = 5) having < 8% normal catalytic activity into the PVN bilaterally. Five days post-injection, the baseline mean arterial pressure in conscious rats was similar in both groups: control, 130 +/- 5 mmHg versus mutant, 122 +/- 6 mmHg. The latency of the pressor response observed after lateral cerebral ventricle injection of 10 pmol ET-1 was 4.8 minutes in controls compared with < 1.5 minutes in rats injected with the mutant nNOS (P < 0.05). After ET-1 administration, the average rise in mean arterial pressure was significantly higher in the nNOS mutant group at 1-2 minutes (16.2 +/- 3.5 mmHg versus -0.6 +/- 4.1 mmHg; P < 0.05) as well as 7-10 minutes later (20.2 +/- 5.1 mmHg versus 8 +/- 2.5 mmHg; P < 0.05). Plasma AVP increased from 2.9 +/- 0.7 pg/mL to 11.5 +/- 1.9 pg/mL in controls (P < 0.004) versus 0.3 +/- 0.2 pg/mL to 1.5 +/- 0.9 pg/mL in the mutant group after ET-1. When the residual effect of nitric oxide generated by other nitric oxide synthase isoforms was assessed by injection of 200 microg Nomega-nitro-L-arginine methyl ester bilaterally into the PVN, the mean arterial pressure increased by 12.2 +/- 2.7 mmHg in controls but was almost unchanged in the mutant group (1.8 +/- 2.4 mmHg; P < 0.025 versus control). These results are consistent with the hypothesis that nitric oxide generated by nNOS within the PVN mediates the inhibition of the pressor response to lateral cerebral ventricle ET-1 and that the greater pressor response seen with the dominant-negative nNOS contructs prevents the rise in plasma AVP in baroreflex-intact rats.

Elevated systemic levels of endothelin-1 and blood pressure correlate with blunted constrictor responses and downregulation of endothelin(A), but not endothelin(B), receptors in an animal model of hypertension.

Hypertension is a major risk factor in the development of cardiovascular disease. Adenovirus gene transfer of endothelin-1 (Ad.CMV.ET-1) in rats produced significant (5-fold) increases in plasma ET-1 and systemic blood pressure (46%) 4 days after viral administration, compared with beta-galactosidase (Ad.CMV.beta-gal) injected as control. The density (B(max)) of the ET receptor ET(A) measured in aortas was reduced significantly by more than 50% to 17+/-2 fmol.mg(-1) of protein for the Ad.CMV.ET-1 group compared with 39+/-6 fmol x mg(-1) of protein for the control. There was no change in the density of the smaller population of the ET(B) sub-type. In agreement, the ratio of ET(A) mRNA to cyclophilin mRNA (a housekeeping gene) measured by Northern analysis was reduced in Ad.CMV.ET-1 rats compared with controls. The ratio of mRNA encoding the ET(B) sub-type did not change. ET-1 vasoconstriction was significantly reduced (P<0.05) in aortas from Ad.CMV.ET-1-treated rats [pD(2)=8.67+/-0.14 (where pD(2) is -log(10)EC(50)); n=11] versus the control (pD(2)=9.11+/-0.06; n=14) but there was no significant difference in the potency of two other vasoconstrictors tested (noradrenaline and Arg-vasopressin), indicating this was a specific effect on ET receptors. There was no change in the affinity of ET-1 binding to either receptor sub-type in the experimental group compared with the control, demonstrating that the attenuation in the constrictor response is the result of the reduced density of receptors rather than a change in affinity. The results show that ET(A) (but not ET(B)) receptors are modulated in this experimental model of hypertension and provide further evidence for selective blockade of the ET(A) receptor as a therapeutic strategy.

Biochemical mechanism of lipid-induced impairment of glucose-stimulated insulin secretion and reversal with a malate analogue.

Hyperlipidemia appears to play an integral role in loss of glucose-stimulated insulin secretion (GSIS) in type 2 diabetes. This impairment can be simulated in vitro by chronic culture of 832/13 insulinoma cells with high concentrations of free fatty acids, or by study of lipid-laden islets from Zucker diabetic fatty rats. Here we show that impaired GSIS is not a simple result of saturation of lipid storage pathways, as adenovirus-mediated overexpression of a cytosolically localized variant of malonyl-CoA decarboxylase in either cellular model results in dramatic lowering of cellular triglyceride stores but no improvement in GSIS. Instead, the glucose-induced increment in "pyruvate cycling" activity (pyruvate exchange with tricarboxylic acid cycle intermediates measured by (13)C NMR), previously shown to play an important role in GSIS, is completely ablated in concert with profound suppression of GSIS in lipid-cultured 832/13 cells, whereas glucose oxidation is unaffected. Moreover, GSIS is partially restored in both lipid-cultured 832/13 cells and islets from Zucker diabetic fatty rats by addition of a membrane permeant ester of a pyruvate cycling intermediate (dimethyl malate). We conclude that chronic exposure of islet beta-cells to fatty acids grossly alters a mitochondrial pathway of pyruvate metabolism that is important for normal GSIS.