The effect of progesterone on prolactin stimulation of fatty acid synthesis, glycerolipid synthesis and lipogenic-enzyme activities in mammary glands of pseudopregnant rabbits, after explant culture or intraductal injection.

The activities of lipogenic enzymes, such as acetyl-CoA carboxylase, fatty acid synthetase and glucose-6-phosphate dehydrogenase, and glycerolipid synthesis increased significantly in mammary explants of 11-day-pseudopregnant rabbits in response to prolactin, in the presence of near-physiological concentrations of insulin and corticosterone in culture. Increasing the concentration of progesterone in culture resulted in suppression of glycerolipid synthesis and activities of acetyl-CoA carboxylase and fatty acid synthetase, but not the pentose phosphate dehydrogenases. However, at near-physiological concentration of progesterone, only acetyl-CoA carboxylase activity was decreased. Injection of prolactin intraductally into 11-day-pseudopregnant rabbits stimulated glycerolipid synthesis, fatty acid synthesis and enzymes involved in fatty acid synthesis, after 3 days. Intraductal injection of progesterone separately or together with prolactin had no significant effect on basal or stimulated lipogenesis in mammary glands. Intramuscular injection of progesterone at 10 mg/day did not suppress fatty acid synthesis stimulated when prolactin was injected intraductally, but a significant inhibition was observed at a higher dose (80 mg/day).     

Changes in the sensitivity of lipogenesis in rat hepatocytes to hormones and precursors over the diurnal cycle and during longer-term starvation of donor animals

In rat hepatocytes freshly isolated from donor rats at different times of the day, the rates of lipogenesis (de novo fatty acid synthesis) varied with a diurnal periodicity. The maximal rate occurred approximately 5 hr after the end of the normal 8-hr feeding period and at this time was four- to fivefold higher than the minimum rate which occurred midway through the feeding period. A similar diurnal pattern of change persisted even when the supply of lipogenic substrate, present in the medium as pyruvate, was not limiting. Although insulin stimulated the basal rates of lipogenesis to different relative extents in hepatocytes isolated at different times of the day, in absolute terms the hormone had little effect on the overall pattern of change during the diurnal cycle. The presence of pyruvate protected lipogenesis against inhibition by glucagon. The degree of protection varied over the diurnal cycle. During the early stages of starvation (up to 24 hr) there was a continuous decline in the rate of hepatocyte lipogenesis, irrespective of whether insulin and/or lipogenic substrate (pyruvate) were available or not. After this time the decline in the rate of lipogenesis was much less rapid. Seventeen hr after removal of food from donor rats, a point was reached beyond which pyruvate was incapable of supporting the maximum basal rate of lipogenesis which occurred during the normal diurnal cycle of fed rats. After this time lipogenesis in the presence of pyruvate was inhibited by glucagon to a much greater relative extent than that observed during feeding. The results suggest that variations in the rate of lipogenesis over the diurnal cycle and during the first 24 hr of starvation could not be accounted for entirely by fluctuations in substrate availability. In contrast, changes which occurred subsequent to this (up to 43 hr of starvation) could be eliminated when lipogenic substrate was made more abundant. Longer periods of starvation were marked by a relative increase in the ability of glucagon to prevent the substrate-induced stimulation of lipogenesis.     

Incorporation of glucose into lipid of perirenal and subcutaneous adipocytes of rats and sheep: influence of insulin

Adipocytes were prepared by collagenase digestion of perirenal and subcutaneous fat from rats and sheep and were incubated in vitro with various concentrations of glucose and insulin. The lipogenic rate of perirenal and subcutaneous adipocytes of rats showed a quadratic response to glucose concentration. The addition of 10 nM insulin increased lipogenesis, especially at low lipogenic rates. At constant glucose concentrations, insulin concentrations up to 50 nM stimulated lipogenesis a similar amount in adipocytes from both depots. The rate of lipogenesis increased relative to cell volume in perirenal adipocytes only. The lipogenic rate of perirenal and subcutaneous adipocytes of sheep showed a positive linear response to glucose concentration, but insulin did not affect the rate of lipogenesis in adipocytes from either depot. In both rats and sheep, the rate of lipogenesis was higher in the perirenal adipocytes. It was concluded that insulin is unlikely to be the agent responsible for the differential growth rates of subcutaneous and perirenal fat depots in rats or sheep.     

Temporal Synergism of Corticosterone and Prolactin in the Syrian Hamster, Mesocricetus auratus

To augment the limited work reported in the literature regarding testing of the hormonal temporal synergism hypothesis in Syrian hamsters (Joseph MM, Meier AH. Proc Soc Exp Biol Med. 1974;146:1150-5), a large experiment with female hamsters was conducted. Forty-eight received corticosterone at 18:00 h on January 21, 23, 25, 27, and 29 and ovine prolactin at one of six times of day beginning January 22 for 8 days; 36 received saline (at 18:00) and prolactin at one of the six times of day for 8 days; 35 received only prolactin at one of the six times of day for 8 days; and 16 received no injections. Twelve hamsters receiving corticosterone and prolactin and eight uninjected hamsters were on running wheels. The corticosterone and prolactin group not on wheels had a body weight gain and no circadian rhythm of weight gain, but did have circadian rhythms of response in organ weight, per 100 g of body weight, and in weights of fat pads and uteri. The corticosterone and prolactin group with access to running wheels gained in body weight and had larger ovaries and smaller fat pads. Hamsters receiving saline and prolactin had a body weight gain, but had no circadian rhythms of response in organ weights. The hamsters receiving only prolactin gained in body weight but had no rhythms of response, except for unexpected circadian rhythms in body weight gain and weights of fat pads. The uninjected hamsters had a modest weight gain. Most or all hamsters with access to running wheels freeran, and the corticosterone injections did not appear to synchronize the locomotor activity rhythms. In conclusion, corticosterone does interact with the injection time effect of prolactin on weights of fat pads, paired ovaries, and uteri. The mechanism of that effect, in terms of circadian rhythm theory, is unclear.     

Cancer Cells Differentially Activate and Thrive on De Novo Lipid Synthesis Pathways in a Low-Lipid Environment

Increased lipogenesis is a hallmark of a wide variety of cancers and is under intense investigation as potential antineoplastic target. Although brisk lipogenesis is observed in the presence of exogenous lipids, evidence is mounting that these lipids may adversely affect the efficacy of inhibitors of lipogenic pathways. Therefore, to fully exploit the therapeutic potential of lipid synthesis inhibitors, a better understanding of the interrelationship between de novo lipid synthesis and exogenous lipids and their respective role in cancer cell proliferation and therapeutic response to lipogenesis inhibitors is of critical importance. Here, we show that the proliferation of various cancer cell lines (PC3M, HepG2, HOP62 and T24) is attenuated when cultured in lipid-reduced conditions in a cell line-dependent manner, with PC3M being the least affected. Interestingly, all cell lines - lipogenic (PC3M, HepG2, HOP62) as well as non-lipogenic (T24) - raised their lipogenic activity in these conditions, albeit to a different degree. Cells that attained the highest lipogenic activity under these conditions were best able to cope with lipid reduction in term of proliferative capacity. Supplementation of the medium with very low density lipoproteins, free fatty acids and cholesterol reversed this activation, indicating that the mere lack of lipids is sufficient to activate de novo lipogenesis in cancer cells. Consequently, cancer cells grown in lipid-reduced conditions became more dependent on de novo lipid synthesis pathways and were more sensitive to inhibitors of lipogenic pathways, like Soraphen A and Simvastatin. Collectively, these data indicate that limitation of access to exogenous lipids, as may occur in intact tumors, activates de novo lipogenesis is cancer cells, helps them to thrive under these conditions and makes them more vulnerable to lipogenesis inhibitors. These observations have important implications for the design of new antineoplastic strategies targeting the cancer cell''s lipid metabolism.     

A diurnal rhythm of pituitary prolactin activity with diurnal effects of mammalian and teleostean prolactin on total body lipid deposition and liver lipid metabolism in teleost fishes*

Previous investigations have shown that mammalian prolactin has a pronounced effect on lipid deposition in teleost fishes (e.g. Meier et al., 1971; de Vlaming & Sage, 1972). Prolactin can be either lipogenic or lipolytic depending on when during the day it is injected. Studies were initiated to determine whether the diurnal effects of prolactin on lipid metabolism in Fundulus similis vary with season or photoperiod acclimation. Data presented here indicate that the diurnal rhythm of response to prolactin persists in the pre-spawning and post-spawning seasons if F. similis is maintained on a long photoperiod. When animals are acclimated to a short photoperiod, treatment with mammalian prolactin either early or late in the photoperiod causes lipid depletion. Homogenates of the portion of the pituitary containing only prolactin cells cause lipid depletion in F. similis when injected early during a long photoperiod, but stimulate lipogenesis if injected late in the photoperiod; these results imply that fish prolactin may be involved in lipid metabolism. Furthermore, prolactin activity in the pituitary of F. similis varies diurnally. In vitro studies of lipid metabolism in Notemigonous crysoleucas and Ictalurus natalis suggest that the liver may be one of the target organs for prolactin.     

Temporal Synergism of Corticosterone and Prolactin in the Syrian Hamster,Mesocricetus auratus

To augment the limited work reported in the literature regarding testing of the hormonal temporal synergism hypothesis in Syrian hamsters (Joseph MM, Meier AH. Proc Soc Exp Biol Med. 1974;146:1150-5), a large experiment with female hamsters was conducted. Forty-eight received corticosterone at 18:00 h on January 21, 23, 25, 27, and 29 and ovine prolactin at one of six times of day beginning January 22 for 8 days; 36 received saline (at 18:00) and prolactin at one of the six times of day for 8 days; 35 received only prolactin at one of the six times of day for 8 days; and 16 received no injections. Twelve hamsters receiving corticosterone and prolactin and eight uninjected hamsters were on running wheels. The corticosterone and prolactin group not on wheels had a body weight gain and no circadian rhythm of weight gain, but did have circadian rhythms of response in organ weight, per 100 g of body weight, and in weights of fat pads and uteri. The corticosterone and prolactin group with access to running wheels gained in body weight and had larger ovaries and smaller fat pads. Hamsters receiving saline and prolactin had a body weight gain, but had no circadian rhythms of response in organ weights. The hamsters receiving only prolactin gained in body weight but had no rhythms of response, except for unexpected circadian rhythms in body weight gain and weights of fat pads. The uninjected hamsters had a modest weight gain. Most or all hamsters with access to running wheels freeran, and the corticosterone injections did not appear to synchronize the locomotor activity rhythms. In conclusion, corticosterone does interact with the injection time effect of prolactin on weights of fat pads, paired ovaries, and uteri. The mechanism of that effect, in terms of circadian rhythm theory, is unclear.     

Role of pyruvate carboxylase in fatty acid synthesis: Alterations during preadipocyte differentiation

Transport of mitochondrial acetyl units to the cytoplasm for fatty acid synthesis via the citrate cleavage pathway requires replenishment of mitochondrial oxaloacetate. Pyruvate carboxylase is though to fulfill this role although compelling evidence has been lacking. During lipogenic differentiation of 3T3-L1 preadipocytes, pyruvate carboxylase activity rises 18-fold in close coordination with fat accumulation and the activity of ATP-citrate lyase, an established lipogenic enzyme. The activities of enzymes less directly related to lipogenesis rise only 3–5-fold while other unrelated enzymes do not increase significantly. These results indicate that pyruvate carboxylase is in fact a lipogenic enzyme.     

Influence of plane of winter nutrition on plasma concentrations of prolactin and testosterone and their association with voluntary food intake in red deer stags (Cervus elaphus)

Blood samples were taken at weekly or 3-weekly intervals from 12 red deer stags, from 3 weeks of age until 3 years of age. Six of the stags were fed to appetite throughout the study and six were maintained on 70% as much during each winter and to appetite each summer. All plasma samples were analysed for testosterone and prolactin. Food intake was measured daily. Annual rhythms of testosterone, prolactin and food intake were evident. Peaks of prolactin, on an increasing photoperiod tended to occur slightly before peaks of food intake and troughs of food intake on a decreasing photoperiod occurred at or slightly after peaks of testosterone. The reduced plane of winter nutrition tended to delay rather than alter the amplitude of the annual cycles of testosterone, prolactin or food intake. Several of the unrestricted stags showed biannual rhythms of testosterone, prolactin and food intake accompanied by additional cycles of coat development and antler growth. These additional cycles tended to be 6 months out of rhythm with normal cycles. It is considered that this is due to the stags being stimulated by a particular day-length irrespective of whether it is decreasing or increasing and that plane of nutrition may influence the control of phase blockers.     

The control of lipogenesis by dietary linoleic acid and its influence on the deposition of fat.

The replacement of dietary starch by sucrose results in an increase in hepatic lipogenesis in the rat. When corn oil (4% by weight or 9% of the energy content of the diet) was included with the sucrose (20% by weight, 20% of the energy content) the lipogenic effect of the sucrose was completely suppressed. In contrast, when beef tallow replaced the corn oil, the induced activity caused by the sucrose was reduced by only approximately 20%. No significant differences were observed between males and females. These diets containing sucrose supplemented with either 4% (w/w) corn oil or 4% (w/w) beef tallow, were then used to ascertain whether or not the effects on hepatic lipogenesis were reflected in changes in the amount of fat deposited during growth from 4--24 weeks of age. It was shown that the percentage body fat was only statistically different (P less than 0.05) when animals fed sucrose-supplemented diets were compared with animals fed diets supplemented with sucrose and beef tallow. However, there were no significant differences in total carcass weight of these rats. The results are discussed in terms of the relative contribution of liver and adipose tissue to total lipogenesis and the factors which control the lipogenic activity in the two tissues.     

Development of fatty acid-synthetic capacity in interscapular brown adipose tissue during suckling in genetically obese Zucker rats.

The development of the lipogenic capacity in brown adipose tissue was studied in suckling lean (Fa/fa) and obese (fa/fa) Zucker pups aged from 7 to 22 days. In both lean and obese pups, activities of the two key lipogenic enzymes, fatty acid synthetase and acetyl-CoA carboxylase, and of citrate cleavage enzyme rose from the early to the late suckling period. Compared with lean pups, 7-day-old fa/fa pups showed a 35% increase in fat accumulation in interscapular brown adipose tissue and a 25% increase in fatty acid synthetase activity. By 10 days of age, fat deposition, lipogenesis in vivo (assessed by the incorporation of 3H from 3H2O into fatty acids) and fatty acid synthetase activity were 1.5-2-fold higher in pre-obese than in lean pups. Compared with lean pups, the increased lipogenesis in vivo observed in brown adipose tissue of 10-day-old pre-obese pups could not entirely account for the difference in fat deposition observed in this tissue, suggesting that additional mechanisms are at play to explain the increased fat content of this tissue.     

Comparative studies of tidal rhythms. VI. Several clocks govern the activity of two species of fiddler crabs

The tide‐associated persistent activity rhythms of the ocypod crabs, Uca pugnax and U. pugilator were observed in constant conditions, some for as long as three months. In some animals the twice/day peaks of activity were found to scan the day at significantly different rates from one another. Individual peaks in some animals split into two fragments. And at times, one peak would fade and then return, or vanish completely. These examples of the independence between tide‐related activity peaks have become the basis for a hypothesis that each peak is controlled by its own clock(s). The basic period of these clocks is “circalunidian”, with periods varying up to about 4–8% on either side of 24.8 hours.     

Circadian and circatidal rhythms of oxygen consumption in the sandy-beach isopod Tylos granulatus Krauss

The sandy-beach isopod Tylos granulatus Krauss burrows at the high tide mark, and has rhythms of nocturnal emergence coincident with the low tide period. Measurements of the respiration rate show that a low rate of oxygen consumption is maintained throughout the day but there is a circatidal rhythmic increase of between 300 and 700% during the nocturnal low tide. The height of this peak and the percentage of animals displaying a peak both increase from spring tide to neap tide suggesting a semi-lunar rhythm. The respiratory peaks have a 24.8 h periodicity, being later each night until low tide falls in the dawn, when there is a ‘switch-back’ so that peaks are then during the subsequent evening low tide. These respiratory rhythms are persistent under constant light and coincide with previously described activity rhythms. The respiratory rhythms considerably reduce metabolic energy losses, particularly as the activity rhythms ensure avoidance of high diurnal temperatures and activity during the cooler nocturnal period.     

Rhythmic changes in the activities of NAD kinase and NADP phosphatase in the achlorophyllous ZC mutant of Euglena gracilis Klebs (strain Z)

NAD kinase and NADP phosphatase activities were detected in the supernatant and the pellet fractions prepared by sonication and centrifugation of the achlorophyllous ZC mutant of the phytoflagellate Euglena gracilis. A detailed study of substrate concentration-velocity curves enabled us to define the saturating substrate concentrations that were used in the enzyme assays. An analysis of the reproducibility of the entire assay procedure indicated that the pooled standard error was about 14%. We report circadian variations in the activities of NAD kinase and NADP phosphatase in the soluble and membrane-bound fractions of both synchronously dividing and nondividing cultures maintained in constant darkness. Bimodal circadian rhythms in total NADP phosphatase activity were found in dividing cells (peaks at circadian times [CT] 00 and 12). The peak observed at CT 00-03 disappeared when the cells had ceased dividing, a result that suggests that it might be regulated by the cell division cycle. NAD kinase activity displayed unimodal circadian rhythms (peak at CT 12) in dividing cells, which persisted with the same phase after the culture entered the stationary phase of growth. Results are discussed with reference to a model (K. Goto, D. L. Laval-Martin, and L. N. Edmunds, Jr., 1985, Science 228, 1284-1288) in which we have proposed that the Ca2(+)-transport system, Ca2+, calmodulin, NAD kinase, and NADP phosphatase could represent clock "gears" that might constitute a self-sustained circadian oscillating loop.     

Effect of thyroidectomy on rhythmic gonadotropin release.

Nonstress blood samples were obtained from intact and thyroidectomized (TE) male rats at 3-hr intervals over a 24-hr period via rapid decapitation. The animals were thyroidectomized when 40 days old and used 6 weeks later. Intact animals showed periodicity in serum LH (P less than 0.01) and prolactin (P less than 0.01). Both gonadotropins began increasing after 8 PM and peak levels occurred at 11 PM. In contrast, 24-hr periodicity was not observed in serum FSH. Corticosterone levels in these same serum samples showed the expected circadian periodicity. After TE, the 24-hr pattern in all gonadotropins was altered significantly. Serum LH increased (P less than 0.01) and circadian periodicity appeared to be absent. FSH and prolactin levels were increased and decreased, respectively (P less than 0.01), with serum prolactin showing a 9-hr phase shift. Prolactin began increasing at 2 AM and reached a peak at 8 AM. Corticosterone in TE animals showed a 24-hr rhythm similar to that of intact rats. These findings confirm our previous observations that nonstress serum LH and prolactin levels fluctuate with a 24-hr periodicity and suggest that the level of, and the phase angle betweeen, these rhythms is markedly influenced by pituitary-thyroid activity.     

Circadian Neuroendocrine Role in Age-Related Changes in Body Fat Stores and Insulin Sensitivity of the Male Sprague-Dawley Rat

A role for circadian neuroendocrine rhythms in the age-related development of obesity and insulin resistance was investigated in the male Sprague-Dawley rat. The phases and amplitudes of the plasma rhythms of several metabolic hormones (i.e. corticosterone, prolactin, insulin, and triiodothyronine) differed in lean, insulin-sensitive (3-week-old rats). insulin-resistant (8-week-old rats) and obese, insulin-resistant (44-week-old rats) animals. Simulation of the daily rhythms of endogenous corticosterone and prolactin by daily injections of the hormones at times corresponding to the peak levels found in 3-week-old rats reversed age-related increases in insulin resistance and body fat in older (5-6-month-old) rats. Ten such daily injections of corticosterone and prolactin in 12-14-week-old rats produced long-term reductions in body fat stores (30%). plasma insulin concentration (40%'). and insulin resistance (60%) (determined by a glucose tolerance test) measured 11-14 weeks after the treatment. Alterations in circadian neuroendocrine rhythms may account for age-related changes in carbohydrate and lipid metabolism in the male Sprague-Dawley rat, and resetting of these rhythms by appropriately timed daily injections of corticosterone and prolactin may help maintain metabolism characteristic of younger animals.     

Circadian Neuroendocrine Role in Age-Related Changes in Body Fat Stores and Insulin Sensitivity of the Male Sprague-Dawley Rat

A role for circadian neuroendocrine rhythms in the age-related development of obesity and insulin resistance was investigated in the male Sprague-Dawley rat. The phases and amplitudes of the plasma rhythms of several metabolic hormones (i.e. corticosterone, prolactin, insulin, and triiodothyronine) differed in lean, insulin-sensitive (3-week-old rats). insulin-resistant (8-week-old rats) and obese, insulin-resistant (44-week-old rats) animals. Simulation of the daily rhythms of endogenous corticosterone and prolactin by daily injections of the hormones at times corresponding to the peak levels found in 3-week-old rats reversed age-related increases in insulin resistance and body fat in older (5-6-month-old) rats. Ten such daily injections of corticosterone and prolactin in 12-14-week-old rats produced long-term reductions in body fat stores (30%). plasma insulin concentration (40%″). and insulin resistance (60%) (determined by a glucose tolerance test) measured 11-14 weeks after the treatment. Alterations in circadian neuroendocrine rhythms may account for age-related changes in carbohydrate and lipid metabolism in the male Sprague-Dawley rat, and resetting of these rhythms by appropriately timed daily injections of corticosterone and prolactin may help maintain metabolism characteristic of younger animals.     

Effect of Carbohydrate Overfeeding on Whole Body and Adipose Tissue Metabolism in Humans

Objective: To evaluate the effect of a 4‐day carbohydrate overfeeding on whole body net de novo lipogenesis and on markers of de novo lipogenesis in subcutaneous adipose tissue of healthy lean humans. Research Methods and Procedures: Nine healthy lean volunteers (five men and four women) were studied after 4 days of either isocaloric feeding or carbohydrate overfeeding. On each occasion, they underwent a metabolic study during which their energy expenditure and net substrate oxidation rates (indirect calorimetry), and the fractional activity of the pentose‐phosphate pathway in subcutaneous adipose tissue (subcutaneous microdialysis with 1, 6¹³C₂, 6, 6²H₂ glucose) were assessed before and after administration of glucose. Adipose tissue biopsies were obtained at the end of the experiments to monitor mRNAs of key lipogenic enzymes. Results: Carbohydrate overfeeding increased basal and postglucose energy expenditure and net carbohydrate oxidation. Whole body net de novo lipogenesis after glucose loading was markedly increased at the expense of glycogen synthesis. Carbohydrate overfeeding also increased mRNA levels for the key lipogenic enzymes sterol regulatory element‐binding protein‐1c, acetyl‐CoA carboxylase, and fatty acid synthase. The fractional activity of adipose tissue pentose‐phosphate pathway was 17% to 22% and was not altered by carbohydrate overfeeding. Discussion: Carbohydrate overfeeding markedly increased net de novo lipogenesis at the expense of glycogen synthesis. An increase in mRNAs coding for key lipogenic enzymes suggests that de novo lipogenesis occurred, at least in part, in adipose tissue. The pentose‐phosphate pathway is active in adipose tissue of healthy humans, consistent with an active role of this tissue in de novo lipogenesis.     

Insulin action on adipocytes. Evidence that the anti-lipolytic and lipogenic effects of insulin are mediated by the same receptor.

1. The dose-response relationships of insulin stimulation of lipogenesis and inhibition of lipolysis were studied simultaneously by using rat adipocytes to determine whether these different effects of insulin are mediated through the same or different sets of receptors. 2. The sensitivity (defined as the concentration of insulin required to produce a half-maximal effect) of the stimulated lipogenic response to insulin was not significantly different from the sensitivity of the anti-lipolytic response to insulin. The addition of different adrenaline and glucose concentrations did not alter the half-maximal concentration of insulin required to inhibit lipolysis. 3. The specificities of the lipogenic and antilipolytic responses were studied by using insulin analogues. The sensitivities of the lipogenic and anti-lipolytic responses were the same for five chemically modified insulins and hagfish insulin, which have potencies compared with bovine insulin of between 3 and 90%. 4. Starving rats for 48h significantly increased the sensitivities of both the antilipolytic and lipogenic responses to insulin, but the changes in the sensitivities of both lipogenesis and anti-lipolysis returned to that of fed rats. 5. We conclude that insulin stimulates lipogenesis and inhibits lipolysis over the same concentration range. These observations provide powerful evidence that the different effects of insulin are mediated through the same set of receptors.