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H Huang, K Rajkumar and L J Murphy

Abstract

The hepatic and renal expressions of the insulin-like growth factor binding proteins (IGFBPs) were examined in transgenic (Tg) mice which overexpress a rat IGFBP-1 transgene driven by the phosphoglycerate kinase-1 promoter. There were no significant differences in the abundance of serum IGFBPs in Tg and wild-type (Wt) mice. Although total hepatic IGFBP-1 mRNA (mouse and transgene-derived) levels were similar in Tg mice to the levels of mouse IGFBP-1 mRNA in Wt mice on day 1 of life, in Tg mice only ∼30% of the IGFBP-1 mRNA was derived from transcription of the mouse gene. An age-related decline in hepatic IGFBP-1 mRNA levels was apparent in both Tg and Wt mice. Food deprivation resulted in increased levels of mouse IGFBP-1 mRNA but the total IGFBP-1 mRNA levels were not significantly different in Tg and Wt mice. In the kidney, unlike the liver, IGFBP-1 mRNA levels in Tg mice were markedly elevated compared with Wt mice and no significant decline was seen with age. Northern blots of hepatic and renal RNA demonstrated similar levels of IGFBP-3, -4, -5 and -6 mRNAs in Tg and Wt mice. From these data we can conclude that in the liver expression of the transgene leads to a coordinated reduction in mouse IGFBP-1 mRNA levels.

Journal of Endocrinology (1997) 152, 99–108

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K Rajkumar, T Modric and LJ Murphy

Differentiation of precursor cells into mature fat cells is accompanied by enhanced expression of insulin-like growth factor (IGF)-I and is stimulated by multiple hormones including growth hormone, glucocorticoids, IGF-I and insulin. We used transgenic mice that overexpress insulin-like growth factor binding protein-1 to investigate the role of IGF-I in the accumulation of fat tissue. In response to a sucrose-enriched diet, transgenic mice gained significantly less body weight and the epididymal fat mass was significantly reduced compared with wild-type mice. The increase in adipocyte size was also significantly reduced in transgenic mice compared with wild-type mice. Fewer colonies were generated from adipose tissue from transgenic mice and the mitogenic response of these cells to IGF-I was significantly reduced compared with those from wild-type mice. Induction of glycerol-3-phosphate dehydrogenase, a measure of adipocyte differentiation, by IGF-I but not insulin, was reduced in preadipocytes from transgenic mice. These data indicate that IGF-I has a critical role in the proliferation of adipocyte precursors, the differentiation of preadipocytes and the development of obesity in response to calorie excess.

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ST Dheen, K Rajkumar and LJ Murphy

Transgenic mice which overexpress insulin-like growth factor binding protein-1 (IGFPB-1) demonstrate fasting hyperglycemia, hyperinsulinemia and glucose intolerance in adult life. Here we have examined the ontogeny of pancreatic endocrine dysfunction and investigated islet cell proliferation and apoptosis in this mouse model. In addition we have examined pancreatic insulin content in transgenic mice derived from blastocyst transfer into non-transgenic mice. Transgenic mice were normoglycemic at birth but had markedly elevated plasma insulin levels, 56.2 +/- 4.5 versus 25.4 +/- 1.5 pmol/l, p < 0.001, and pancreatic insulin concentration, 60.5 +/- 2.5 versus 49.0 +/- 2.6 ng/mg of tissue, P < 0.01, compared with wild-type mice. Transgenic mice derived from blastocyst transfer to wild-type foster mothers had an elevated pancreatic insulin content similar to that seen in pups from transgenic mice. There was an age-related decline in pancreatic insulin content and plasma insulin levels and an increase in fasting blood glucose concentrations, such that adult transgenic mice had significantly less pancreatic insulin than wild-type mice. Pancreatic islet number and the size of mature islets were increased in transgenic animals at birth compared with wild-type mice. Both islet cell proliferation, measured by 5-bromo-2'-deoxyuridine labeling, and apoptosis, assessed by the in situ terminal deoxynucleotidyl transferase and nick translation assay, were increased in islets of newborn transgenic mice compared with wild-type mice. In adult mice both islet cell proliferation and apoptosis were low and similar in transgenic and wild-type mice. Islets remained significantly larger and more numerous in adult transgenic mice despite a reduction in pancreatic insulin content. These data suggest that overexpression of IGFBP-1, either directly or indirectly via local or systemic mechanisms, has a positive trophic effect on islet development.

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K. Rajkumar, P. J. Chedrese, H. Ly and B. D. Murphy

ABSTRACT

LH, in addition to increasing cyclic AMP (cAMP) in ovarian cells, stimulates phosphoinositide hydrolysis producing inositol trisphosphate and diacylglycerol (DG). DG activates phospholipid- and calcium-dependent protein kinase (PKC). In the present study, we have used both PKC activators and inhibitors to examine the interactions of the PKC pathway on hormone-induced cAMP production in porcine luteal cells. Phorbol 12-myristate 13-acetate (PMA) enhanced LH- and forskolin-induced cAMP production. A time-course study indicated that the facilitatory effect of PMA was greater when added to incubation tubes following addition LH or forskolin. The non-tumour-promoting phorbol ester 4α-phorbol 12,13-didecanoate, which does not stimulate PKC activation, did not facilitate hormone-induced cAMP induction. PKC inhibitors polymyxin B, sphingosine and 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H7) antagonized the facilitatory effect of PMA on LH-induced cAMP production. The cAMP induction by both LH and forskolin was inhibited in the presence of PKC inhibitors. Polymyxin E, which differs from polymyxin B by a single amino acid and does not inhibit PKC activation, did not inhibit LH- or forskolin-induced cAMP induction.

The results of this study provide evidence for a facilitative action of the PKC effector system on hormonally stimulated cAMP production. Furthermore, PKC may be an important endogenous regulator of adenylate cyclase activity in porcine luteal cells.

Journal of Endocrinology (1991) 130, 273–280

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K. Rajkumar, D. E. Kerr, R. N. Kirkwood and B. Laarveld

ABSTRACT

Somatostatin-14 (SRIF-14) inhibited, in a concentration-dependent manner, LH- and forskolin-stimulated cyclic adenosine monophosphate (cAMP) induction in porcine granulosa and luteal cells. The inhibitory effect of SRIF-14 on hormone-induced cAMP generation was more potent in porcine ovarian cells than in the GH-3 pituitary cell line. The inhibitory effect of SRIF-14 was impeded by neutralizing its biological activity with specific antiserum. Preincubation of luteal and granulosa cells with phorbol 12-myristate 13-acetate (PMA) enhanced LH- and forskolin-stimulated cAMP levels. SRIF-14 failed to inhibit LH- or forskolin-stimulated cAMP levels in cells preincubated with PMA. It is concluded that SRIF-14 inhibits hormone-stimulated cAMP induction in the porcine ovary. LH-induced protein kinase C activation may be physiologically important to alleviate the inhibitory effects of SRIF-14.

Journal of Endocrinology (1992) 134, 297–306

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K. Rajkumar, H. Ly, P. W. Schott, B. Njaa and B. D. Murphy

ABSTRACT

The present studies were carried out to compare the low density lipoprotein (LDL) metabolism by freshly isolated immature porcine granulosa cells with that by luteal cells. Furthermore, we have examined the effect of serum used for plating of granulosa cells on lipoprotein degradation and utilization. In incubation studies, addition of LDL as an exogenous substrate had a mild stimulatory effect on progesterone accumulation by granulosa cells, while it exhibited a dose-dependent stimulatory effect on luteal cells. When granulosa and luteal cells were incubated with 125I-labelled LDL, membrane binding of LDL occurred in both cell types, but only luteal cells were capable of internalizing the bound LDL. Granulosa cells in incubation degraded LDL much less in comparison with luteal cells, and the amount varied with the maturity of the cells. When granulosa cells were plated with graded amounts of serum which was withdrawn for 48 h following plating, they exhibited enhanced LDL degradation in a serum concentration-dependent fashion. Addition of serum for plating selectively enhanced utilization of LDL, but not high density lipoprotein (HDL) for progesterone accumulation by the cells in culture. Time-course studies on LDL degradation by granulosa cells following serum withdrawal indicate that the ability of cells to degrade LDL decreased in a time-dependent fashion. Serum withdrawal selectively decreased utilization of LDL but not HDL for progesterone secretion. It is concluded that immature granulosa cells have a limited capability to utilize cholesterol carried by LDL. However, when cultured in the presence of serum, cells acquire the ability to utilize more efficiently LDL-carried cholesterol for progesterone secretion which is then lost following long-term withdrawal of serum from culture.

Journal of Endocrinology (1989) 122, 557–564