Laxmi S. Srivastava

Glucocorticoids are required for food deprivation-induced increases in hypothalamic neuropeptide Y expression

Neuropeptide Y (NPY), a 36 amino‐acid peptide found within the hypothalamus, is thought to be an important regulator of food intake. Hypothalamic NPY gene expression, synthesis and secretion are all known to be increased in models of increased metabolic demand in which serum glucocorticoids are also elevated. The present studies were designed to test the hypothesis that glucocorticoids are required for increased hypothalamic preproNPY mRNA levels induced by food deprivation (FD). First, animals underwent bilateral sham‐adrenalectomy (sham) or not (control), and were subjected to 72 h FD, or not. Total RNA was isolated from hypothalamic tissue blocks and the content of preproNPY mRNA was measured by solution hybridization/RNase protection analysis. This study revealed that there was no significant difference in hypothalamic preproNPY mRNA content between shamfed and control‐fed groups, or between sham‐FD and control‐FD groups. In the second experiment, animals underwent bilateral adrenalectomy (ADX), were allowed to feed ad libitum and were sacrificed 1 day, 4 days and 7 days after ADX. Nuclease protection analysis revealed no significant effect of ADX on hypothalamic preproNPY mRNA levels over this time‐course. Finally, we examined the role of glucocorticoids in regulating NPY gene expression following FD. Animals underwent bilateral ADX, or not. At the time of surgery, ADX animals received placebo, or corticosterone (B) replacement in the form of constant release pellets, at one of two doses. Food was removed from half of the animals in each group 24 h after surgery; all animals were sacrificed 72 h thereafter. There was no difference in preproNPY mRNA content between the ADX‐FD and ADX‐fed groups, relative to the fed controls. Replacement with corticosterone [ADX(B)] did not alter preproNPY mRNA content in fed animals, however preproNPY mRNA content in FD animals was increased 2.5‐fold. These studies demonstrate that glucocorticoids are necessary and serve a stimulatory role in the increase in hypothalamic preproNPY mRNA levels observed under conditions of FD, and suggest that hypothalamic NPY gene expression may be directly responsive to peripheral metabolic and hormonal signals.

Apolipoprotein CII and lipoprotein lipase in human nephrotic syndrome

Plasma apolipoprotein (apo CII), apo CII in plasma very low density lipoproteins (VLDL), plasma post-heparin lipoprotein lipase (LPL) activity and urinary apo CII excretion were assessed in 9 patients with nephrotic syndrome. Apo CII comprised a significantly lower percentage of plasma VLDL protein (mean ± SEM) in nephrotic patients (7.2 ± 0.4%) than normals (10 ± 0.5%), but was similar to levels in patients with primary hypertriglyceridemia and normal renal function (7.4 ± 0.6%). The ability of VLDL from nephrotic patients to activate lipoprotein lipase in vitro (79.8 ± 14.1 units/mg protein and 18.2 ± 3.6 units/M triglyceride TG) was significantly lower than normal (158 ± 11.1 units mg protein and 36.8 ± 4.4 units/M TG) but was similar to primary hypertriglyceridemics cer.0 ± 8.1 units/mg protein and 17.6 ± 2.2 units/M TG). Whereas in primary hypertriglyceridemics the amount of apo CII/mg VLDL protein or M TG was inversely proportional to (log10) total plasma TG, such a relationship was not observed in nephrotic patients. Post-heparin plasma hepatic lipase was significantly lower in male nephrotics (2.9 ± 2.5 μM FFA/ml/h) compared to normals (14.4 ± 4.2). Hepatic LPL correlated inversely with total plasma low density lipoprotein cholesterol (r = −0.49, P < 0.05). Post-heparinnon-hepatic lipase was low in 2 and low normal in 2 of 8 patients studied. Mean 24-h urinary excretion of apo CII was higher in nephrotic patients (241 ± 116 μg) than normals (27 ± 5 μg) and primary hypertriglyceridemics (31 ± 5 μg). Total plasma apo CII in nephrotics (89.1 ± 14.0 μg/ml) was higher than normal (51.8 ± 3.2 μg/ml; P < 0.01) and similar to patients with primary type IIB (89.1 ± 4.6 μg/ml) and primary type IV hyperlipoproteinemia (85.4 ± 6.9 μg/ml). Six of 9 nephrotic patients had 24-h urinary excretion rates of apo CII which were within the normal range while 3 patients had markedly increased apo CII excretion. These studies suggest that in nephrotic syndrome, (a) the decreased LPL activator potency in VLDL may reflect reduced apo CII per VLDL particle, or an excess of LPL inhibitory apolipoproteins and (b) decreased hepatic and extra-hepatic hepoprotein lipase levels may relate to altered lipoprotein catabolism and (c) increased urinary loss of apo CII does not result in its depletion in plasma.

C-II anapolipoproteinemia and severe hypertriglyceridemia: Report of a rare case with absence of C-II apolipoprotein isoforms and review of the literature

A new case of C-II anapolipoproteinemia (complete apolipoprotein C-II deficiency) as the cause of severe hypertriglyceridemia with chylomicronemia (type I lipoprotein phenotype) is described. The patient was a five-year-old boy living in Connecticut. He had splenomegaly, episodic abdominal pain, and bloody stools. Absence of apolipoprotein C-II (and its isoforms C-II1 and C-II2) was documented by a sensitive and specific radioimmunoassay, analytical isoelectric focusing, and in vitro lipolytic assay. Decreased levels of high- and low-density lipoprotein cholesterol and apolipoproteins A-I and A-II and increased levels of plasma triglycerides and apolipoprotein E were found. Post-heparin extra-hepatic lipoprotein lipase activity was within normal range. Incorporation of exogenous purified human apolipoprotein C-II to an incubation mixture of purified lipoprotein lipase and the patients triglyceride-rich lipoproteins resulted in a dramatic increase in the catabolic rate of the defective triglyceride-rich lipoproteins. The absence of the isoforms of apolipoprotein C-II in this patient indicates that a common gene exists for the C-II isoproteins, which appear to be necessary for normal triglyceride transport in humans. A literature review of 23 reported cases indicates that xanthomas and hepatosplenomegaly are less common in C-II anapolipoproteinemia than in lipoprotein lipase deficiency, the other major etiologic cause of genetic chylomicronemia.

The role of high density lipoprotein apolipoprotein CII in triglyceride metabolism.

The purpose of these studies was (a) to examine the relationship between total plasma triglycerides (TG) and the amount of apolipoprotein CII (apo CII) in triglyceride rich lipoproteins (TRL), and (b) to determine whether TRL could be enriched with apo CII in vitro. In 13 patients with primary endogenous hypertriglyceridemia, (log10) total plasma TG correlated inversely with the amount of apo CII per unit very low density lipoprotein (VLDL) protein (r=−0.76;p<0.005) and VLDL TG (r=−0.75; p<0.005). The potency of VLDL to activate milk lipoprotein lipase (LPL) in hydrolyzing triolein was studied in vitro. LPL activator potency per unit VLDL protein or VLDL TG correlated inversely with (log10) total plasma TG (r=−0.86 and r=−0.76, respectively; p<0.005). LPL activator potency per nM VLDL apo CII also correlated inversely with (log10) total plasma TG (r=−0.49; p<0.01). In seven patients with familial type V hyperlipoproteinemia, the average amount of apo CII in TRL protein was subnormal (5.86±0.62% vs 10.0±0.51% in normal subjects). The higher the (log10) total plasma TG, the lower was the apo CII content in TRL protein (r=−0.93; p<0.01). To determine the factors governing the distribution of apo CII between lipoproteins and whether TRL could be enriched with apo CII, five approaches were undertaken: (a)125I apo CII was added to mixtures of VLDL and HDL. The amount of labelled apo CII in VLDL was proportional to the ratio of VLDL to HDL. (b) TRL from four patients with familial type V hyperlipoproteinemia was incubated with high density lipoprotein (HDL) from a normal subject. An increase in the TRL/HDL ratio was associated with transfer of apo CII from HDL to TRL and a reciprocal transfer of non-apo CII protein from TRL to HDL. Net apo CII enrichment of TRL protein was possible below a HDL/TRL protein ratio of ca. 6 under the experimental conditions. (c) A fixed amount of normal plasma feed of TRL was incubated with different amounts of TRL from two patients with familial type V hyperlipoproteinemia. The amount of apo CII that transferred from normal TRL free plasma to the patient’s TRL was proportional to the amount of TRL in the mixture. (d) A doubling and tripling in the amount of apo CII in TRL was found when apo CII was added directly to TRL from a normal subject and TRL from a patient with familial type V hyperlipoproteinemia, respectively. (e) When apo CII was added directly to normal plasma and plasma from a patient with primary type IV hyperlipoproteinemia, the peptide was taken up mainly by VLDL and HDL, indicating enrichment of these fractions. The distribution of the added apo CII in each lipoprotein fraction resembled the distribution in the native plasma. TRL was isolated after addition of apo CII to plasma from two patients with familial types IV and V, respectively. Enrichment of TRL with apo CII was associated with an approximate 1.5-fold increase in the LPL activator potency per unit TRL protein. These studies suggest that firstly, the amount of apo CII in TRL is inversely related to the severity of hypertriglyceridemia. Secondly, the distribution of apo CII between TRL and HDL is governed by the mass ratios of these two lipoprotein classes. Thirdly, plasma TRL and HDL have a reserve binding capacity of apo CII and fourthly, it is possible to enrich these lipoproteins with this functionally important peptide. Whether net enrichment of TRL with apo CII and also an increase in its biological activity to activate LPL in vitro is related to increased in vivo catabolic rate requires to be determined.

Immunological studies on bovine milk lipoprotein lipase. Effects of Fab fragments on enzyme activity.

Rabbit antiserum was prepared against purified bovine mild lipoprotein lipase. Immunoelectrophoresis of lipoprotein lipase gave a single precipitin line against the antibody which was coincident with enzyme activity. The gamma-globulin fraction inhibited heparin-releasable lipoprotein lipase activity of bovine arterial intima, heart muscle and adipose tissue. The antibody also inhibited the lipoprotein lipase activity from adipose tissue of human and pig, but not that of rat and dog. Fab fragments were prepared by papain digestion of the gamma-globulin fraction. Fab fragments inhibited the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine vesicles and trioleoylglycerol emulsions to the same extent. The Fab fragments also inhibited the lipolysis of human plasma very low density lipoproteins. The change of the kinetic parameters for the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol by the Fab fragments was accompanied with a 3-fold increase in Km and a 10-fold decrease in Vmax. Preincubation of lipoprotein lipase with apolipoprotein C-II, the activator protein for lipoprotein lipase, did not prevent inhibition of enzyme activity by the Fab fragments. However, preincubation with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol or Triton X-100-emulsified trioleoylglycerol had a protective effect (remaining activity 7.0 or 25.8%, respectively, compared to 1.0 or 0.4% with no preincubation). The addition of both apolipoprotein C-II and substrate prior to the incubation with the Fab fragments was associated with an increased protective effect against inhibition of enzyme activity; remaining activity with dipalmitoylphosphatidylcholine-emulsified trioleoylglycerol was 40.6% and with Triton X-100-emulsified trioleoylglycerol, 45.4%. Human plasma very low density lipoproteins also protected against the inhibition of enzyme activity by the Fab fragments. These immunological studies suggest that the interaction of lipoprotein lipase with apolipoprotein C-II in the presence of lipids is associated with a conformational change in the structure of the enzyme such that the Fab fragments are less inhibitory. The consequence of a conformational change in lipoprotein lipase may be to facilitate the formation of an enzyme-triacylglycerol complex so as to enhance the rate of the lipoprotein lipase-catalyzed turnover of substrate to products.

Plasma aldosterone concentrations in neonatal spontaneously hypertensive rats

Abstract Plasma aldosterone concentrations were found to be higher in the newborn, presumably prehypertensive, spontaneously hypertensive rat at 5, 10 and 20 days of age compared to age-matched normotensive Wistar-Kyoto (WKY). In addition, plasma aldosterone concentrations were seen to rise in newborn WKY in contrast to findings in Wistar rats and other mammalian species.

Effect of a high carbohydrate diet on the content of apolipoproteins C-II, C-III and E in human plasma high density lipoprotein subfractions

The effect of isocaloric high and low carbohydrate (Carb) diets on the structure and apoprotein composition of plasma high density lipoproteins (HDL) was assessed in four healthy men. The high Carb diet contained 65% calories as Carb and 15% as fat; the low Carb was 15% and 65%, respectively, with protein fixed at 20% of calories in each case. Cholesterol was 400 mg/day and the P/S ratio of the fat was 0.4. Each diet was sequentially consumed for periods of 3 weeks. At the end of each 3-week study period, plasma HDL2 and HDL3 were isolated by zonal ultracentrifugation and their apoprotein and lipid compositions were determined. Compared to the low Carb diet, the high Carb diet was associated with an increase in the size of HDL2 (116.0 +/- 1.8 vs. 109.1 +/- 1.8 A) and in the content (mean weight % +/- SEM) of apoE (2.81 +/- 0.71 vs. 1.79 +/- 0.49, P less than 0.01) and of apoC-II (1.73 +/- 0.09 vs. 1.11 +/- 0.12, P less than 0.01). HDL2 apoC-III content was not significantly different on the two diets (6.49 +/- 0.50 vs. 7.42 +/- 1.21). On the two diets, HDL3 size and HDL3 apoE content were not significantly changed. HDL3 apoC-II and apoC-III, however, were higher on the high Carb diet, P less than 0.05. The ratio (by weight) of HDL2 apoE/HDL2 apoC-II + C-III increased on the high Carb diet compared to the low Carb diet (0.344 +/- 0.058 vs. 0.228 +/- 0.053, P less than 0.01). We suggest that the increased amount of apolipoprotein E in HDL2 may influence its rate of catabolic clearance and may account for the well-known decrease in plasma HDL-cholesterol in subjects on high Carb diets.