Effects of maternal nutrient restriction, intrauterine growth restriction, and glucocorticoid exposure on phosphoenolpyruvate carboxykinase-1 expression in fetal baboon hepatocytes in vitro

Background: The objective of this study was to develop a cell culture system for fetal baboon hepatocytes and to test the hypotheses that (i) expression of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase-1 (PEPCK-1) is upregulated in hepatocytes isolated from fetuses of nutrient-restricted mothers (MNR) compared with ad libitum-fed controls (CTR), and (ii) glucocorticoids stimulate PEPCK-1 expression. Methods: Hepatocytes from 0.9G CTR and MNR fetuses were isolated and cultured. PEPCK-1 protein and mRNA levels in hepatocytes were determined by Western blot and quantitative PCR, respectively. Results: Fetuses of MNR mothers were intrauterine growth restricted (IUGR). Feasibility of culturing 0.9G fetal baboon hepatocytes was demonstrated. PEPCK-1 protein levels were increased in hepatocytes isolated from IUGR fetuses, and PEPCK-1 mRNA expression was stimulated by glucocorticoids in fetal hepatocytes. Conclusions: Cultured fetal baboon hepatocytes that retain their in vivo phenotype provide powerful in vitro tools to investigate mechanisms that regulate normal and programmed hepatic function. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Geldanamycin Derivative Ameliorates High Fat Diet-Induced Renal Failure in Diabetes

Diabetic nephropathy is a serious complication of longstanding diabetes and its pathogenesis remains unclear. Oxidative stress may play a critical role in the pathogenesis and progression of diabetic nephropathy. Our previous studies have demonstrated that polyunsaturated fatty acids (PUFA) induce peroxynitrite generation in primary human kidney mesangial cells and heat shock protein 90β1 (hsp90β1) is indispensable for the PUFA action. Here we investigated the effects of high fat diet (HFD) on kidney function and structure of db/db mice, a widely used rodent model of type 2 diabetes. Our results indicated that HFD dramatically increased the 24 h-urine output and worsened albuminuria in db/db mice. Discontinuation of HFD reversed the exacerbated albuminuria but not the increased urine output. Prolonged HFD feeding resulted in early death of db/db mice, which was associated with oliguria and anuria. Treatment with the geldanamycin derivative, 17-(dimethylaminoehtylamino)-17-demethoxygeldanamycin (17-DMAG), an hsp90 inhibitor, preserved kidney function, and ameliorated glomerular and tubular damage by HFD. 17-DMAG also significantly extended survival of the animals and protected them from the high mortality associated with renal failure. The benefit effect of 17-DMAG on renal function and structure was associated with a decreased level of kidney nitrotyrosine and a diminished kidney mitochondrial Ca2+ efflux in HFD-fed db/db mice. These results suggest that hsp90β1 is a potential target for the treatment of nephropathy and renal failure in diabetes

Moderate Global Reduction in Maternal Nutrition Has Differential Stage of Gestation Specific Effects on β1- and β2-Adrenergic Receptors in the Fetal Baboon Liver

Hepatic β-adrenergic receptors (β-ARs) play a pivotal role in mobilization of reserves via gluconeogenesis and glycogenolysis to supply the animal with its energy needs during decreased nutrient availability. Using a unique nutrient-deprived baboon model, we have demonstrated for the first time that immunoreactive hepatic β1- and β2-AR subtypes are regionally distributed and localized on cells around the central lobular vein in 0.5 and 0.9 gestation (G) fetuses of ad libitum fed control (CTR) and maternal nutrient restricted (MNR) mothers. Furthermore, MNR decreased fetal liver immunoreactive β1-AR and increased immunoreactive β2-AR at 0.5G. However, at 0.9G, immunohistochemistry and Western blot analysis revealed a decrease in β1-AR and no change in β2-AR levels. Thus, MNR in a nonhuman primate species has effects on hepatic β1- and β2-ARs that are receptor- and gestation stage-specific and may represent compensatory systems whose effects would increase glucose availability in the presence of nutrient deprivation

Impact of 17-DMAG on hsp90β1, [Ca²⁺]_m, and peroxynitrite generation in the kidney.

<p>Western blot analysis was performed to assess hsp90β1 in kidney homogenate (<i>A</i>) and isolated mitochondria (<i>B</i>) of HFD-fed <i>db/db</i> mice with 6 animals per group. Linoleic acid (LA)-induced [Ca²⁺]_m efflux and peroxynitrite generation (<i>C–F</i>) in kidney mitochondria were measured.</p

Effect of 17-DMAG on survival rate of HFD-fed <i>db/db</i> mice.

<p>Kaplan-Meyer survival analysis was performed using the log-rank statistics to measure the difference between the survival curves of d<i>b/db-HF-S</i> vs <i>db/db-HF-G</i> mice with n = 9 per group. Parallel experiments were performed with <i>db/+</i> mice (<i>db/+-HF-S</i> and <i>db/+-HF-G</i> groups) and no mortality was observed.</p

Effects of high fat diet (HFD) and 17-DMAG treatment on kidney function of <i>db/db</i> mice.

<p>(<i>A</i>) Schematic of two phases of HFD feeding and 17-DMAG treatment with the arrows indicating the scheduled kidney function assessments. During the first phase of HFD and the subsequent regular diet (RD) feeding, the 24 h urinary albumin excretion (<i>B</i>) and urine output (<i>C</i>) were measured to assess kidney functions in <i>db/db</i> and the non-diabetic control (<i>db/+</i>) mice. During the second phase of HFD feeding, the animals were either injected with saline (<i>HF-S</i>) or 17-DMAG (<i>HF-G</i>) and the kidney functions were initially assessed by the 24 h urinary albumin excretion (<i>D</i>) and urine output (<i>E</i>), and then by serum creatinine (<i>G</i>) when anuria or oliguria occurred. Blood glucose was measured as indicated in (<i>F)</i>. **<i>P</i><0.01, compared with baseline assessed on day 0 with n = 6–12 per group.</p