The effect of chronic insulin delivery via the intraperitoneal versus the subcutaneous route on hepatic triglyceride secretion rate in streptozotocin diabetic rats.

Chronic intraperitoneal or subcutaneous insulin administration increases triglyceride secretion rate (TGSR) in normal rats. We wished to determine the effect of this treatment on TGSR and the hepatic lipogenic enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) in diabetic rats. Streptozotocin-diabetic rats, untreated (D), diabetic rats treated with insulin (3 U/day for 21 days) intraperitoneally (IP) or subcutaneously (SC) and non-diabetic rats (N) were studied. TGSR was determined using Triton WR-1339. Fasting glucose and triglyceride levels, high in D, were normalized by insulin treatment regardless of route. Peripheral insulin levels were lowest in D and highest in SC, portal insulin levels were lowest in D and highest in IP. Non-esterified fatty acid levels were not elevated in D, presumably due to adipose tissue depletion. TGSR was reduced in D (P<0.05) and was normalized following insulin administration, regardless of route. ACC activity was normal, but FAS was decreased in D (P<0.05). ACC and FAS were normal in both IP and SC. Thus, in streptozotocin-diabetic rats, chronic intraperitoneal or subcutaneous insulin treatment increases TGSR and FAS activity from their low levels in insulin-deficient rats to levels equal to but not higher than those in normal rats.

Chronic intraperitoneal insulin delivery, as compared with subcutaneous delivery, improves hepatic glucose metabolism in streptozotocin diabetic rats.

We have previously shown that chronic insulin treatment by the intraperitoneal route normalizes the elevated glucose production (GP) in streptozotocin (STZ) diabetic rats, while insulin delivered by the subcutaneous route only partially normalizes GP. To investigate the biochemical mechanism of the effect of chronic insulin delivery by either route on hepatic glucose metabolism, we measured the hepatic activity of glucose 6-phosphatase (G6Pase) and glucokinase (GK). Four groups of rats were used: (1) nondiabetic rats (N, n = 7), (2) untreated STZ diabetic rats (D, n = 8), (3) diabetic rats treated intraperitoneally (IP, n = 6), or (4) subcutaneously (SC, n = 8) (both 3 U of insulin/d). Glucose levels, higher in D, were normalized by insulin treatment regardless of route. Peripheral insulin levels were lowest in D and highest in SC as expected (N, 162 +/- 18 pmol/L; D, 66 +/- 12; IP, 360 +/- 96; SC, 798 +/- 198). STZ diabetes resulted in a 10-fold decrease in GK (P < .001), and a 2-fold increase in G6Pase activity (P < .01). Both intraperitoneal and subcutaneous treatments normalized G6Pase activity. In contrast, with subcutaneous but not intraperitoneal treatment, GK activity was still 35% less than normal (SC v N, P < .05). Glucose 6-phosphate (G6P) levels did not differ among the groups. In summary: (1) the increase in GP in D reflected increased activity of G6Pase and reduced activity of GK, (2) the partial suppression of GP with subcutaneous insulin treatment reflected correction of increased G6Pase activity, but only partial correction of low GK activity, and (3) the normalization of GP with intraperitoneal insulin treatment reflected correction of both increased G6Pase activity and low GK activity. Our current studies indicate that chronic intraperitoneal insulin treatment is superior to subcutaneous treatment with regard to hepatic glucose metabolism.

Prolonged elevation of plasma free fatty acids desensitizes the insulin secretory response to glucose in vivo in rats.

Prolonged exposure of pancreatic islets to free fatty acids (FFAs) inhibits glucose-stimulated insulin secretion (GSIS) in vitro. However, FFA inhibition of GSIS has not been clearly demonstrated in vivo. We examined the in vivo effect of prolonged elevation of plasma FFAs on GSIS using a two-step hyperglycemic clamp in rats treated with a 48-h intravenous infusion of either 20% Intralipid plus heparin (INT) (5 microl/min plus heparin, 0.1 U/min; n = 8), oleate (OLE) (1.3 microEq/min; n = 6), saline (SAL) (n = 6), or bovine serum albumin (BSA) (vehicle for OLE; n = 5). Because there was no difference in any of the parameters between BSA and SAL rats, these groups were combined as control rats (CONT) (n = 11). At the end of the 48-h OLE/INT/CONT infusions, after an overnight fast, plasma glucose was clamped for 2 h at 13 mmol/l and for another 2 h at 22 mmol/l. Preclamp plasma FFAs were elevated twofold (P < 0.01) versus CONT with both INT and OLE (NS, INT vs. OLE). Preclamp glucose, insulin, and C-peptide levels were higher in INT than in CONT rats (P < 0.05), suggesting insulin resistance, but they were not different in OLE and CONT rats. The insulin and C-peptide responses to the rise in plasma glucose from basal to 13 mmol/l were lower in OLE (336 +/- 72 pmol/l and 1.2 +/- 0.1 nmol/l, P < 0.01 and P < 0.05, respectively) than in CONT (552 +/- 54 and 1.9 +/- 0.1) rats, but they were not different between CONT and INT rats (648 +/- 150 and 2.0 +/- 0.4). The insulin and C-peptide responses to the rise in plasma glucose from 13 to 22 mmol/l were lower in both INT (1,188 +/- 204 pmol/l and 3.0 +/- 0.3 nmol/l, P < 0.01 and P < 0.001) and OLE (432 +/- 60 and 1.7 +/- 0.2, P < 0.001 vs. CONT or INT) rats than in CONT rats (1,662 +/- 174 and 5.0 +/- 0.6). In summary, 1) both INT and OLE decreased GSIS in vivo in rats, and 2) the impairing effect of INT on GSIS was less than that of OLE, which might be due to the different type of fatty acid (mostly polyunsaturated in INT versus monounsaturated as OLE) and/or to differential effects of INT and OLE on insulin sensitivity. In conclusion, prolonged elevation of plasma FFAs can desensitize the insulin secretory response to glucose in vivo, thus inducing a beta-cell defect that is similar to that found in type 2 diabetes.

The role of factors that regulate the synthesis and secretion of very-low-density lipoprotein by hepatocytes.

Lipoproteins are particles that contribute to overall metabolic homeostasis by transporting hydrophobic lipids in the blood plasma to and from different tissues in the body. Very-low-density lipoprotein (VLDL) is the principal vehicle for the transport of endogenous triglyceride (TG), and, ultimately, through its metabolic product, low-density lipoprotein (LDL), of cholesterol as well. It is synthesized mainly in hepatocytes, with small amounts also being produced by enterocytes in the fasting state. The mechanism of VLDL assembly is complex and is regulated at different levels by a variety of factors. The main structural protein of VLDL is called apolipoprotein B-100 (Apo B). Apo B formation and degradation therefore represent two major points of regulation of VLDL secretion. Hepatic levels of lipids such as phosphatidylcholine (PC), cholesteryl ester (CE), fatty acids (FA), and TG also affect VLDL synthesis. There are different views as to the specific mechanism by which each lipid class affects VLDL particle formation. In general, PC appears to promote the translocation of apo B from the cytosol to the lumen of the endoplasmic reticulum, a step that is crucial in the early stages of VLDL assembly. Apo B degradation is suppressed, and therefore VLDL secretion is enhanced, in the presence of elevated CE levels. For TG to be incorporated into the lipoprotein, it requires the action of a protein called microsomal triglyceride transfer protein (MTP). MTP might have a preference for TG comprised of FA with a certain degree of saturation. It becomes apparent that changes in diet that are accompanied by variations in the type of fats that are ingested affect VLDL formation and secretion. Regulation also occurs post-prandially in response to elevations in plasma insulin levels. Acute elevations in insulin inhibit VLDL secretion by promoting the degradation of apo B. This action is consistent with insulin's anabolic properties as it allows for the hepatic storage of lipid rather than for its distribution in VLDL to other tissues for fuel. Many studies have attempted to unravel the mechanisms of VLDL formation and secretion. The fact that so many factors are involved complicates the issue. The purpose of this article is to describe the relationship between different factors involved in VLDL assembly and secretion so that a better understanding of its metabolic regulation may be achieved.

The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus.

Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.

Mechanisms and management of amphotericin B-induced nephrotoxicity.

PURPOSE: The author outlines the care of patients receiving intravenous amphotericin B, with emphasis on the prevention and/or management of nephrotoxicity. OVERVIEW: The immunocompromised patient remains at risk for systemic fungal infections; however, therapeutic options are limited. Although amphotericin B has remained the drug of choice for more than 30 years, its toxic effects, particularly nephrotoxicity, warrant careful attention. Nephrotoxicity is the most serious and dose-limiting effect of amphotericin B therapy. Appropriate assessment before, during, and after therapy in patients receiving intravenous amphotericin B may help to minimize the potential for nephrotoxicity. CLINICAL IMPLICATIONS: To provide optimal patient care, it is imperative that the clinician understand the etiology of and the signs and symptoms associated with nephrotoxicity, as well as interventions to prevent nephrotoxicity, in the patient receiving amphotericin B.

Characterization of regulatory volume decrease in the THP-1 and HL-60 human myelocytic cell lines.

Exposure to hypotonic stress produces a transient increase in cell volume followed by a regulatory volume decrease (RVD) in both THP-1 and HL-60 cells. In contrast, cells exposed to hypotonic stress in a high K/low Na Hanks' solution not only failed to volume regulate, but displayed a secondary swelling. Thus, while an outward K gradient was required for RVD, the secondary swelling indicated that hypotonic stress increased permeability in the absence of a negative membrane potential. The K channel blocker quinine (1-4 mM) blocked RVD in both cell types. Gramicidin's ability to overcome the quinine block of RVD indicated that RVD is mediated by a quinine-sensitive cation transport mechanism that is independent of the swelling-induced anion transport mechanism. Barium (1-4 mM), another K channel blocker, slowed the rate of RVD, while 4-aminopyridine, charybdotoxin, tetraethylammonium chloride, tetrabutylammonium chloride, and gadolinium had no effect on RVD. Furthermore, RVD was not mediated by calcium-activated conductances, since it occurred normally in Ca-free medium, in medium containing cadmium, and in BAPTA-loaded cells. Gramicidin produced little or no volume change in isotonic medium, suggesting that basal C1 permeability of both THP-1 and HL-60 cells is low. However, swelling induced an anion efflux pathway that is permeable to both chloride and bromide, but is impermeable to methanesulfonate and glutamate. The anion channel blocker 3,5-diiodosalicylic acid (DISA) antagonized RVD in both cell types. In conclusion, RVD in THP-1 and HL-60 cells is mediated by independent anion and cation transport mechanisms that involve both a DISA-sensitive anion pathway and a quinine-inhibitable K efflux pathway, neither of which requires increases in intracellular calcium to be activated.

Alpha-irradiation of haemopoietic tissue in pre- and postnatal mice: 2. Effects of mid-term contamination with 239Pu in utero.

The distribution of 239Pu in various tissues of foetal and postnatal offspring of pregnant mice, injected i.v. at 13 days gestation with 30 kBq 239Pu/kg (in some cases with 10 or 100 kBq/kg), together with the numbers of haemopoietic progenitors in the bone marrow, spleen and liver, were measured through to 1 year post-partum. The quality of the haemopoietic microenvironment in these mice was also measured using the renal-capsule implant method. The largest radiation dose received by any haemopoietic organ was that in the liver, amounting to 10-14 mGy, as reported previously. In spite of normal numbers of haemopoietic spleen colony-forming cells (CFC-S) in the liver and seeding, at birth, into the bone marrow where the level of plutonium was minimal, a long-term deficit in their number rapidly developed. The development of the stromal microenvironment, however, was also deficient, suggesting that the dose of alpha-irradiation to the foetal liver was sufficient to cause sublethal damage in those cells destined to become the precursors of the supportive haemopoietic microenvironment in bone marrow and spleen. The results of this study suggest that although the placenta affords significant shielding to the tissues of the developing foetus from maternal contamination, the long-term effects on haemopoiesis are comparable to those in mice contaminated as adults. This further implies that the developing haemopoietic tissues are exquisitely sensitive to 239Pu contamination.

Alpha-particle irradiation of haemopoietic tissue in pre- and postnatal mice. 1: Distribution of plutonium-239 after mid-term contamination.

Pregnant mice (at 13 days gestation) and age-matched controls were injected with 30 kBq 239Pu/kg and the distribution of plutonium in maternal and foetal tissues measured. Approximately 2% of the activity injected into the mother reached each foetus in 24 h, 95% of which was contained in membranes and placenta. The concentration of plutonium in foetal liver was 3 times the average foetal body concentration; both liver and body concentrations in the foetus increased by the end of gestation. Each pup accumulated only 0.01% extra injected activity after 9 days lactation and, as the resulting concentrations in the neonatal skeleton were low, we conclude that the greatest haemopoietic risk to the offspring from mid-term contamination in utero is in the foetal liver (which received an average dose of 10-14 mGy between the time of mid-term contamination and birth). By the end of gestation about one-quarter of the original activity was transferred to foetal tissues from the maternal liver and skeleton. No significant changes in maternal distribution were detected as a result of lactation. The results of this study are discussed, along with a compilation of previously published data.

The development of spatial distributions of CFU-S and in-vitro CFC in femora of mice of different ages.

The radial distributions of spleen colony forming units (CFU-S) and in-vitro colony forming cells (in-vitro-CFC) were measured in the diaphyseal marrow cavity of femora removed from 3-, 5- and 11-week-old mice. The distributions observed in 11-week-old mice confirm earlier findings that the highest concentrations of CFU-S exist near bone surfaces whereas the concentration of in-vitro-CFC increases to a peak value approximately 300 microns from the femoral axis with a low value at the bone surface. The gradients of the distributions in all three age groups are very similar suggesting that spatial organization in marrow is established by 3 weeks at the latest and, as the marrow cavities grow, so the distributions extend into the new space following their respective gradients. The peak of CFU-S concentration at the bone surfaces in all age groups coincides with increased rates of DNA synthesis and a low self-renewal capacity. Conversely, CFU-S nearer the centre of the cavity maintain a low turnover but have a high self-renewal capacity. Measurements made on 1-week-old mice show that the marrow contains a lower average concentration of CFU-S in the femoral cavity compared to older mice. However, these CFU-S have both a high rate of turnover and a high self-renewal capacity. It appears that these better quality CFU-S remain in a central location while the rest of the population ages and expands in association with growing bone regions.

Long-term haemopoietic injury in mice after repeated irradiation: precursor-cell cycling and its regulation.

The cycling rate of haemopoietic stem cells (day 9 CFU-S) and granulocyte-macrophage colony forming cells (GM-CFC) in mouse femora was, in response to reduced numbers, elevated at all times of sampling between 3 weeks and 10 months after 4 repeated doses of 4.5 Gy X-rays (3 wk between doses). The level of a stimulator of CFU-S cycling was also elevated, and this was observed in both axial and marginal regions of the marrow inside the shaft. However, the rate of production of the stimulator was low; lower than previously reported in marrow regenerating after a single dose of 4.5 Gy, indicating damage to the regulatory stromal cells. The distribution of CFU-S across the axial and marginal zones of femoral marrow was changed from that in normal mice, where higher concentrations were found near the bone surface, to a more uniform distribution.