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E Kim, S Sohn, M Lee, J Jung, R D Kineman and S Park

The impact of streptozotocin (STZ)-induced, insulinopenic diabetes on the GH axis of rats and mice differs from study to study, where this variation may be related to the induction scheme, severity of the diabetes and/or the genetic background of the animal model used. In order to begin differentiate between these possibilities, we compared the effects of two different STZ induction schemes on the GH axis of male Sprague–Dawley rats: (1) a single high-dose injection of STZ (HI STZ, 80 mg/kg, i.p.), which results in rapid chemical destruction of the pancreatic β-cells, and (2) multiple low-dose injections of STZ (LO STZ, 20 mg/kg for 5 consecutive days, i.p.), which results in a gradual, autoimmune destruction of β-cells. STZ-treated animals were killed after 3 weeks of hyperglycemia (>400 mg/dl), and in both paradigms circulating insulin levels were reduced to <40% of vehicle-treated controls. HI STZ-treated rats lost weight, while body weights of LO STZ-treated animals gradually increased over time, similar to vehicle-treated controls. As previously reported, HI STZ resulted in a decrease in circulating GH and IGF-I levels which was associated with a rise in hypothalamic neuropeptide Y (NPY) mRNA (355% of vehicle-treated controls) and a fall in GH-releasing hormone (GHRH) mRNA (45% of vehicle-treated controls) levels. Changes in hypothalamic neuropeptide expression were reflected by an increase in immunoreactive NPY within the arcuate and paraventricular nuclei and a decrease in GHRH immunoreactivity in the arcuate nucleus, as assessed by immunohistochemistry. Consistent with the decline in circulating GH and hypothalamic GHRH, pituitary GH mRNA levels of HI STZ-treated rats were 58% of controls. However, pituitary receptor mRNA levels for GHRH and ghrelin increased and those for somatostatin (sst2, sst3 and sst5) decreased following HI STZ treatment. The impact of LO STZ treatment on the GH axis differed from that observed following HI STZ treatment, despite comparable changes in circulating glucose and insulin. Specifically, LO STZ treatment did suppress circulating IGF-I levels to the same extent as HI STZ treatment; however, the impact on hypothalamic NPY mRNA levels was less dramatic (158% of vehicle-treated controls) where NPY immunoreactivity was increased only within the paraventricular nucleus. Also, there were no changes in circulating GH, hypothalamic GHRH or pituitary receptor expression following LO STZ treatment, with the exception that pituitary sst3 mRNA levels were suppressed compared with vehicle-treated controls. Taken together these results clearly demonstrate that insulinopenia, hyperglycemia and reduced circulating IGF-I levels are not the primary mediators of hypothalamic and pituitary changes in the GH axis of rats following HI STZ treatment. Changes in the GH axis of HI STZ-treated rats were accompanied by weight loss, and these changes are strikingly similar to those observed in the fasted rat, which suggests that factors associated with the catabolic state are critical in modifying the GH axis following STZ-induced diabetes.

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Yi Luan, Maxwell E Edmonds, Teresa K Woodruff and So-Youn Kim

Cancer therapy can cause off-target effects including ovarian damage, which may result in primary ovarian insufficiency in girls and premenopausal women. Loss of ovarian follicles within the ovarian reserve leads to ovarian endocrine dysfunction and impaired fertility. Cyclophosphamide (CPA), a commonly used chemotherapeutic and immunosuppressant agent, is a gonadotoxic agent that destroys ovarian cells by crosslinking DNA. To protect the ovary against CPA damage, we sought to precisely map the mechanism by which the ovarian reserve is depleted by CPA. We found that CPA specifically depletes primordial follicles without affecting primary and secondary follicles in three independent murine strains (CD-1, C57BL/6J and BALB/cJ) in vivo. We directly tested the effect of the active metabolite of CPA, 1 μM 4-hydroxyperoxycyclophophamide (4-HC), in vitro and confirmed the loss of primordial oocytes but no change in the number of primary and secondary follicles. We demonstrated that phospho-AKT (p-AKT) and cleaved PARP (cPARP) are present in primordial oocytes 3 days after CPA injection, consistent with the role of these markers as part of the apoptotic cascade. Interestingly, p-AKT positive primordial oocytes co-expressed cPARP. Treatment of animals with specific inhibitors of apoptotic pathway components, ETP46464 and CHK2, blocked 4-HC‒induced DNA damage in vitro. These data suggest that CPA targets primordial germ cells in the ovarian reserve by stimulating apoptosis pathways. Adjuvant therapies to protect primordial germ cells from the off-target effects of CPA may reduce the risk of POI.

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Seung Jin Han, Sung-E Choi, Sang-A Yi, Soo-Jin Lee, Hae Jin Kim, Dae Jung Kim, Hyun Chul Lee, Kwan Woo Lee and Yup Kang

2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) is an activator of glutamate dehydrogenase (GDH), which is a mitochondrial enzyme with an important role in insulin secretion. We investigated the effect of BCH on the high-glucose (HG)-induced reduction in glucose-stimulated insulin secretion (GSIS), the HG/palmitate (PA)-induced reduction in insulin gene expression, and HG/PA-induced β-cell death. We also studied whether long-term treatment with BCH lowers blood glucose and improves β-cell integrity in db/db mice. We evaluated GSIS, insulin gene expression, and DNA fragmentation in INS-1 cells exposed to HG or HG/PA in the presence or absence of BCH. An in vivo study was performed in which 7-week-old diabetic db/db mice were treated with BCH (0.7 g/kg, n=10) and placebo (n=10) every other day for 6 weeks. After treatment, an intraperitoneal glucose tolerance test and immunohistological examinations were performed. Treatment with BCH blocked HG-induced GSIS inhibition and the HG/PA-induced reduction in insulin gene expression in INS-1 cells. In addition, BCH significantly reduced HG/PA-induced INS-1 cell death and phospho-JNK level. BCH treatment improved glucose tolerance and insulin secretion in db/db mice. BCH treatment also increased the ratio of insulin-positive β-cells to total islet area (P<0.05) and reduced the percentage of β-cells expressing cleaved caspase 3 (P<0.05). In conclusion, the GDH activator BCH improved glycemic control in db/db mice. This anti-diabetic effect may be associated with improved insulin secretion, preserved islet architecture, and reduced β-cell apoptosis.

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Jin W Kim, Robert P Rhoads, Nthabisheng Segoale, Niels B Kristensen, Dale E Bauman and Yves R Boisclair

During the transition from pregnancy to lactation, dairy cows experience a 70% reduction in plasma IGF-I. This reduction has been attributed to decreased hepatic IGF-I production. IGF-I circulates predominantly in multi-protein complexes consisting of one molecule each of IGF-I, IGF binding protein-3 and the acid labile subunit (ALS). Recent studies in the mouse have shown that absence of ALS results in accelerated turnover and severely depressed concentration of plasma IGF-I. These observations suggest that reduced plasma ALS could be a second factor contributing to the fall of plasma IGF-I in peri-parturient cows. This possibility has not been studied due to the lack of bovine ALS reagents. To address this, we isolated the bovine ALS cDNA and used its sequence to develop a ribonuclease protection assay (RPA) and a bovine ALS antiserum. Using the RPA, ALS mRNA abundance was approximately fivefold higher in liver than in lung, small intestine, adipose tissue, kidney and heart, but was absent in muscle and brain. The antiserum detected the highest ALS levels in plasma followed by ovarian follicular fluid, lymph and colostrum. A portion of colostrum and follicular fluid ALS appears to be synthesized locally as ALS mRNA was found in mammary epithelial cells and ovarian follicular cells. Finally, we measured plasma ALS in dairy cows during the peri-parturient period (days −35 and +56 relative to parturition on day 0). Plasma ALS dropped by 50% between late pregnancy and the first day of lactation and returned to prepartum levels by day +56. To determine whether this reflected a change in hepatic expression, ALS mRNA was measured in liver biopsies collected on days −35, +3 and +56. ALS mRNA expression was significantly lower on day +3 than on day −35, but recovered completely by day +56. Finally, we examined the ability of GH to increase plasma ALS abundance at selected times before and after parturition (weeks −5, −2, +1 and +5). GH increased plasma ALS at weeks −5, −2 and +5, but not at week +1. Identical effects of GH were seen when the response considered was plasma IGF-I. We conclude that the decline in plasma ALS after parturition is a consequence of hepatic GH resistance and contributes to the associated reduction of plasma IGF-I.

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R P Rhoads, J W Kim, M E Van Amburgh, R A Ehrhardt, S J Frank and Y R Boisclair

Dairy cows enter a period of energy insufficiency after parturition. In liver, this energy deficit leads to reduced expression of the liver-specific GH receptor transcript (GHR1A) and decreased GHR abundance. As a consequence, hepatic processes stimulated by GH, such as IGF-I production, are reduced. In contrast, adipose tissue has been assumed to remain fully GH responsive in early lactation. To determine whether energy insufficiency causes contrasting changes in the GH responsiveness of liver and adipose tissue, six lactating dairy cows were treated for 4 days with saline or bovine GH when adequately fed (AF, 120% of total energy requirement) or underfed (UF, 30% of maintenance energy requirement). AF cows mounted robust GH responses in liver (plasma IGF-I and IGF-I mRNA) and adipose tissue (epinephrine-stimulated release of non-esterified fatty acids in plasma, IGF-I mRNA, and p85 regulatory subunit of phosphatidylinositol 3-kinase mRNA). Reductions of these responses were seen in the liver and adipose tissue of UF cows and were associated with decreased GHR abundance. Reduced GHR abundance occurred without corresponding reductions of GHR1A transcripts in liver or total GHR transcripts in adipose tissue. In contrast, undernutrition did not alter the abundance of proteins involved in the early post-receptor signaling steps. Thus, a feed restriction reproducing the energy deficit of early lactation depresses GH actions not only in liver but also in adipose tissue. It remains unknown whether a similar reduction of GH action occurs in the adipose tissue of early lactating dairy cows.

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D. J. J. Carr, B. R. DeCosta, C.-H. Kim, A. E. Jacobson, V. Guarcello, K. C. Rice and J. E. Blalock

ABSTRACT

Opioid peptides have been shown to modulate various parameters of both the humoral and cellular arms of the immune system. The modulatory capacity of the peptides can often be substantially reduced in the presence of naloxone, an opioid receptor antagonist, indicating a classical ligand-receptor interaction. In order to characterize these interactions further, we investigated the characteristics of opioid receptors on a macrophage cell line, P388d1. A δ-class opioid receptor was found with an M r of 58 000. We also identified opioid receptors on MOLT-4 (T-cell) and IM-9 (B cell) cell lines as well as thymocytes and T celland B cell-enriched populations. Using the central (brain) κ-selective agonist, U-69,593, it was also determined that P388d1 cells possess κ-like opioid receptors. Scatchard analysis of the binding of [3H]U-69,593 revealed a single population of sites with a dissociation constant of 17 ± 3 (s.e.m.) nmol/l and a total number of binding sites of 53·8 ± 1·0 (s.e.m.) fmol/106 cells. Moreover, the racemic κ-selective agonist U-50,488H was able to displace 50% of [3H]U-69,593 binding at 8·0 nmol/l, whereas other opioid ligands such as [Met]-enkephalinamide (δ-selective) and [d-Ala2,N - Me - Phe4,Gly5 - ol] - enkephalin (μ - selective) were ineffective displacers of [3H]U-69,593 except at high concentrations.

Journal of Endocrinology (1989) 122, 161–168

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Iain R Thompson, Annisa N Chand, Kim C Jonas, Jacky M Burrin, Mark E Steinhelper, Caroline P Wheeler-Jones, Craig A McArdle and Robert C Fowkes

In the pituitary, C-type natriuretic peptide (CNP) has been implicated as a gonadotroph-specific factor, yet expression of the CNP gene (Nppc) and CNP activity in gonadotrophs is poorly defined. Here, we examine the molecular expression and putative function of a local gonadotroph natriuretic peptide system. Nppc, along with all three natriuretic peptide receptors (Npr1, Npr2 and Npr3), was expressed in both αT3-1 and LβT2 cells and primary mouse pituitary tissue, yet the genes for atrial-(ANP) and B-type natriuretic peptides (Nppa and Nppb) were much less abundant. Putative processing enzymes of CNP were also expressed in αT3-1 cells and primary mouse pituitaries. Transcriptional analyses revealed that the proximal 50 bp of the murine Nppc promoter were sufficient for GNRH responsiveness, in an apparent protein kinase C and calcium-dependent manner. Electrophoretic mobility shift assays showed Sp1/Sp3 proteins form major complexes within this region of the Nppc promoter. CNP protein was detectable in rat anterior pituitaries, and electron microscopy detected CNP immunoreactivity in secretory granules of gonadotroph cells. Pharmacological analyses of natriuretic peptide receptor activity clearly showed ANP and CNP are potent activators of cGMP production. However, functional studies failed to reveal a role for CNP in regulating cell proliferation or LH secretion. Surprisingly, CNP potently stimulated the human glycoprotein hormone α-subunit promoter in LβT2 cells but not in αT3-1 cells. Collectively, these findings support a role for CNP as the major natriuretic peptide of the anterior pituitary, and for gonadotroph cells as the major source of CNP expression and site of action.

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Holly M Johnson, Erin Stanfield, Grace J Campbell, Erica E Eberl, Gregory J Cooney and Kim S Bell-Anderson

Poor nutrition plays a fundamental role in the development of insulin resistance, an underlying characteristic of type 2 diabetes. We have previously shown that high-fat diet-induced insulin resistance in rats can be ameliorated by a single glucose meal, but the mechanisms for this observation remain unresolved. To determine if this phenomenon is mediated by gut or hepatoportal factors, male Wistar rats were fed a high-fat diet for 3 weeks before receiving one of five interventions: high-fat meal, glucose gavage, high-glucose meal, systemic glucose infusion or portal glucose infusion. Insulin sensitivity was assessed the following day in conscious animals by a hyperinsulinaemic-euglycaemic clamp. An oral glucose load consistently improved insulin sensitivity in high-fat-fed rats, establishing the reproducibility of this model. A systemic infusion of a glucose load did not affect insulin sensitivity, indicating that the physiological response to oral glucose was not due solely to increased glucose turnover or withdrawal of dietary lipid. A portal infusion of glucose produced the largest improvement in insulin sensitivity, implicating a role for the hepatoportal region rather than the gastrointestinal tract in mediating the effect of glucose to improve lipid-induced insulin resistance. These results further deepen our understanding of the mechanism of glucose-mediated regulation of insulin sensitivity and provide new insight into the role of nutrition in whole body metabolism.

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Hyo-Eun Kim, Sung-E Choi, Soo-Jin Lee, Ji-Hyun Lee, Youn-Jung Lee, Sang Sun Kang, Jaesun Chun and Yup Kang

The present study was undertaken to determine how tumour necrosis factor-α (TNF-α) elicits the inhibition of glucose-stimulated insulin secretion (GSIS) in rat insulinoma cells (INS)-1 β-cells. TNF-α pretreatment did not change the expression levels of insulin, PDX-1, glucose transporter 2, glucokinase, KATP channels, Ca2 + channels, and exocytotic molecules and, furthermore, did not reduce the glucose-stimulated ATP level. On the other hand, TNF-α reduced the glucose-stimulated influx of Ca2 +. The TNF-α treatment was thought to activate c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and NF-κB inflammatory signals, since TNF-α increased phospho-JNK and phospho-p38 and reduced IκB levels. Inhibitors of these signaling pathways prevented the TNF-α-induced reduction of the Ca2 + influx and GSIS. Overexpression of MEKK3, a possible mediator from the TNF-α receptor to the JNK/p38 and NK-κB signaling cascade, increased the levels of phospho-JNK, phospho-p38, and NF-κB, and reduced the glucose-stimulated Ca2 + influx and GSIS. The reduction of the Ca2 + influx and GSIS in MEKK3-overexpressing INS-1 cells was also prevented by inhibitors of JNK, p38, and NF-κB. These data demonstrate that TNF-α inhibits GSIS by reducing the glucose-stimulated Ca2 + influx, possibly through the activation of JNK and p38 MAPK and NF-κB inflammatory signals. Thus, our findings suggest that the activation of stress and inflammatory signals can contribute to the inhibition of GSIS in the development of diabetes.

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Daniel J Tobiansky, George V Kachkovski, Reilly T Enos, Kim L Schmidt, E Angela Murphy and Kiran K Soma

Sucrose consumption is associated with type 2 diabetes, cardiovascular disease, and cognitive deficits. Sucrose intake during pregnancy might have particularly prominent effects on metabolic, endocrine, and neural physiology. It remains unclear how consumption of sucrose affects parous females, especially in brain circuits that mediate food consumption and reward processing. Here, we examine whether a human-relevant level of sucrose before, during, and after pregnancy (17–18 weeks total) influences metabolic and neuroendocrine physiology in female rats. Females were fed either a control diet or a macronutrient-matched, isocaloric sucrose diet (25% of kcal from sucrose). Metabolically, sucrose impairs glucose tolerance, increases liver lipids, and increases a marker of adipose inflammation, but has no effect on body weight or overall visceral adiposity. Sucrose also decreases corticosterone levels in serum but not in the brain. Sucrose increases progesterone levels in serum and in the brain and increases the brain:serum ratio of progesterone in the mesocorticolimbic system and hypothalamus. These data suggest a dysregulation of systemic and local steroid signalling. Moreover, sucrose decreases tyrosine hydroxylase (TH), a catecholamine-synthetic enzyme, in the medial prefrontal cortex. Finally, sucrose consumption alters the expression pattern of FOSB, a marker of phasic dopamine signalling, in the nucleus accumbens. Overall, chronic consumption of sucrose at a human-relevant level alters metabolism, steroid levels, and brain dopamine signalling in a female rat model.