J. Dairy Sci. 90:1810-1815. doi:10.3168/jds.2006-631
© American Dairy Science Association, 2007.
Phlorizin Induces Lipolysis and Alters Meal Patterns in Both Early-and Late-Lactation Dairy Cows1
B. J. Bradford2 and
M. S. Allen3
Department of Animal Science, Michigan State University, East Lansing 48824
3 Corresponding author: allenm{at}msu.edu
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ABSTRACT
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Phlorizin is known to increase whole-body glucose demand, but it has also stimulated lipolysis in past studies in ruminants. Increased lipolysis complicates studies of dry matter intake (DMI) regulation by hepatic oxidation by providing the liver with additional oxidative substrate. Therefore, to assess whether increased glucose demand selectively increases DMI for cows in negative energy balance, phlorizin was administered to early- and late-lactation cows. Six Holstein cows in early lactation (19 ± 6 DIM, 50.0 ± 1.8 kg/d of milk, mean ± SD) and 6 Holstein cows in late lactation (228 ± 18 DIM, 30.6 ± 1.9 kg/d of milk) were randomly assigned to treatment sequence in a crossover design. Periods were 14 d with 7-d adaptation periods and 7 d of treatment. Phlorizin (4 g/d) and propylene glycol (carrier and control) were administered subcutaneously every 6 h throughout the treatment periods. Feeding behavior and DMI data were collected for the final 4 d of each treatment period; blood samples and total urine output were collected on d 4 of each treatment period. Phlorizin caused urinary loss of glucose at 333 g/d in early-lactation cows and 532 g/d in late-lactation cows. Phlorizin increased plasma nonesterified fatty acid concentrations similarly in early- and late-lactation cows, but did not significantly alter plasma insulin concentrations. Treatment with phlorizin tended to decrease meal size, but also decreased intermeal interval, resulting in no effect on DMI. The effects of phlorizin on lipolysis, feeding behavior, and DMI are not dependent on relative energy balance.
Key Words: phlorizin • glucose demand • feeding behavior • feed intake
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INTRODUCTION
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Physiological regulation of feed intake must respond to a variety of environmental and endogenous cues to allow growth or maintain BW, support milk production, and supply nutrients for fetal growth. The mechanisms that direct changes in feeding behavior often interact, making it difficult to identify the signals that are most responsible for increased DMI at parturition or following the initiation of bST treatment, for example. Pharmacological treatments that have more specific influences on physiological processes can help identify specific mechanisms that may contribute to the regulation of feed intake.
Phlorizin inhibits renal reabsorption of glucose (Ehrenkranz et al., 2005), resulting in urinary excretion of glucose and a significant increase in whole-body glucose demand. Increasing glucose demand in lactating cows led to an increase in transcript abundance for potentially rate-limiting gluconeogenic enzymes (Bradford and Allen, 2005). We hypothesized that greater gluconeogenic capacity would increase utilization of propionate for glucose production and decrease its oxidation, altering hepatic energy status. Decreased hepatic ATP production may result in delayed satiety during meals, because limiting ATP production by trapping inorganic phosphate increased feed intake in rats (Rawson et al., 1994), as did inhibition of fatty acid (FA) oxidation (Horn et al., 2004). Therefore, we predicted that phlorizin would increase meal size and DMI in lactating cows by limiting propionate oxidation (Allen et al., 2005). However, in previous work, we showed that late-lactation cows were able to adapt to phlorizin administration for 7 d without increasing DMI (Bradford and Allen, 2005). The effects of phlorizin on glucose metabolism were confounded by a concomitant increase in NEFA delivery to the liver, which likely supplied substrate to replace propionate removed from oxidative pathways. We speculated that early-lactation cows, already in negative energy balance, would be less flexible in their use of nutrients to drive gluconeogenesis, and that phlorizin treatment would result in increased DMI in this model. This experiment was designed to test our revised hypothesis.
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MATERIALS AND METHODS
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Experimental procedures were approved by the All-University Committee on Animal Use and Care at Michigan State University.
Design and Treatments
Multiparous Holstein cows were selected from the Michigan State University Dairy Cattle Teaching and Research Center and assigned to blocks of early lactation (n = 6; 19 ± 6 DIM; 2.3 ± 0.5 lactations; 657 ± 68 kg of BW; mean ± SD) and late lactation (n = 6; 228 ± 18 DIM; 2.3 ± 0.8 lactations; 689 ± 77 kg of BW) cows. Stalls were assigned to a block and treatment sequence to ensure balance within blocks, and cows were randomly assigned to stalls within a block. Phlorizin (Sigma Chemical Co., St. Louis, MO) was administered via subcutaneous injection at the rate of 4 g/d, with propylene glycol as the vehicle and control (4 injections/ d, 4 mL/injection). Treatment periods were 7 d, and injections were given every 6 h during these periods. Animals were adapted to a single diet for a 7-d period prior to the first treatment period, and a 7-d rest period was included between the 2 treatment periods. One of the 28 injections during period 2 was missed; however, no data were collected for 36 h after the missed injection, and glucose excretion stabilizes relatively quickly following injection of phlorizin (Amaral-Phillips et al., 1993). Because there were no significant period x treatment interactions for DMI, feeding behavior, or plasma analyte concentrations in subsequent statistical analyses (all P 
0.15), we concluded that the missed injection had little or no effect on measured responses to treatment.
Data and Sample Collection
Throughout the experiment, cows were housed in tie stalls and fed a single experimental diet (Table 1
) as a TMR once daily (1130 h) at 115% of expected daily intake. Cows were not allowed access to feed from 1000 to 1130 h, during which orts and the amount of feed offered were weighed for each cow daily. During the final 4 d of each treatment period, feeding behavior was monitored by a computerized data acquisition system (Dado and Allen, 1993) throughout the day. Data on chewing activities, feed disappearance, and water consumption were recorded for each cow every 5 s, and mean daily values for number of meal bouts, interval between meals, and meal size were calculated. On each of these 4 d, samples of all dietary ingredients (0.5 kg) and orts (12.5%) were collected and frozen for later analysis. Starting on d 4 (1000 h) of each experimental period, urine was collected for a 24-h period and blood was sampled hourly from an indwelling jugular catheter as previously described (Bradford and Allen, 2005). Cows were milked twice daily in the milking parlor during rest periods and in the tie stalls during treatment periods. Milk yield was recorded and samples were taken at each milking during the 4 collection days.
Sample Analysis
Diet ingredients, orts, and fecal samples were dried in a 55°C forced-air oven for 72 h and analyzed for DM concentration. Feed samples were ground with a Wiley mill (1-mm screen, Arthur H. Thomas, Philadelphia, PA) and analyzed for ash, NDF, CP, and starch concentrations as previously described (Bradford and Allen, 2005). Concentrations of all nutrients were expressed as percentages of DM determined from drying at 105°C in a forced-air oven.
Milk samples were analyzed for fat, true protein, and lactose with infrared spectroscopy (AOAC, 1990) by Michigan DHIA (East Lansing, MI). In addition, milk samples from d 7 of each treatment period were analyzed for FA profile by gas chromatography as previously described (Bradford and Allen, 2004). The milk 
9-desaturase index was calculated as the sum of the concentrations of 9-C14:1, 9-C16:1, and 9-C18:1, and cis-9, trans-11 18:2, divided by the sum of these concentrations plus C14:0, C16:0, C18:0, and trans-11 18:1. Plasma samples were composited into a single sample for each cow period for all analyses except NEFA and growth hormone (GH) concentration. Urine and plasma samples were analyzed in duplicate for glucose concentration by the glucose oxidase method (Raabo and Terkildsen, 1960). Plasma samples were analyzed in duplicate using commercial kits to determine concentrations of NEFA [NEFA C-kit, Wako Chemicals USA, Richmond, VA, as modified (McCutcheon and Bauman, 1986)], BHBA (procedure #2440, Stanbio Laboratory, Boerne, TX), insulin (Coat-A-Count, Diagnostic Products Corporation, Los Angeles, CA), and glucagon (Glucagon kit #GL-32K, Linco Research Inc., St. Charles, MO). Plasma GH concentrations were quantified with a double-antibody RIA (Gaynor et al., 1995).
Statistical Analysis
One cow in the late-lactation group decreased DMI by approximately 50% at the beginning of each data collection period, likely because of sensitivity to the chew halter apparatus. Because the cow was an outlier for DMI and plasma analyses were indicative of an unusual catabolic state, this cow was removed from all analyses.
Data were analyzed according to the following mixed-effects model:
where Yijkl is a dependent variable, µ is the overall mean, Pi is the fixed effect of period (i = 1 to 2), Sj is the fixed effect of stage of lactation (j = 1 to 2), Tk is the fixed effect of treatment (k = 1 to 2), Cl is the random effect of cow nested within stage of lactation (l = 1 to 6), STjk is the interaction of stage of lactation and treatment, PTik is the interaction of period and treatment, and eijkl is the residual error. Analysis of plasma NEFA and GH data also included fixed effects of sample time, time x treatment interaction, and time x stage interaction. Urinary glucose and plasma BHBA values were not normally distributed and were log-transformed for analysis; reported means were back-transformed. For all main effects, significance was declared at P 
0.05, and tendencies were declared at P 0.10. For interactions, significance was declared at P 0.10, and tendencies were declared at P 0.15.
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RESULTS AND DISCUSSION
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As expected, urinary glucose excretion increased dramatically with phlorizin administration, from 0.4 to 323 g/d in early lactation and 549 g/d in late lactation (P 0.001, Table 2). The significant treatment x stage of lactation interaction (P 0.01) is likely because of significantly higher plasma glucose concentrations in late lactation. Phlorizin-treated cows in late lactation had a mean glucose concentration of 68.2 mg/dL compared with 59.9 mg/dL for treated early-lactation cows. Because phlorizin inhibits renal glucose reabsorption, greater delivery of glucose to the kidneys in late-lactation cows was expected to result in greater glucose excretion.
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Table 2. Effects of phlorizin and stage of lactation on glucose excretion, blood plasma metabolites and hormones, and feed intake1
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As reported previously (Amaral-Phillips et al., 1993; Overton et al., 1998; Bradford and Allen, 2005), phlorizin treatment increased plasma NEFA concentration (P 0.03), which is indicative of the rate of lipolysis in ruminants (Dunshea et al., 1989). Although Amaral-Phillips and coworkers (1993) reported increased lipolysis in response to phlorizin treatment in cows at 6 wk postpartum, the mean NEFA concentration for the control treatment (181 µEq/L) suggested that the cows were not in a severely catabolic state (Grummer, 1993). Nevertheless, our results in cows with higher plasma NEFA concentrations (278 µEq/L) confirm that phlorizin increases lipolysis during negative energy balance, and further demonstrate that early- and late-lactation cows respond to phlorizin with a comparable increase in lipolysis. We and other researchers have suggested that phlorizin-induced lipolysis is mediated by decreased plasma insulin concentrations (Vranic et al., 1984; Bradford and Allen, 2005); however, in this experiment, we found that phlorizin did not significantly alter plasma insulin concentration or the insulin:glucagon ratio (Table 2
).
We then assessed whether GH was responsible for this lipolytic response, because plasma GH concentrations increase in response to hypoglycemia (De Feo et al., 1989) and GH acts to suppress lipogenesis in ruminant adipose tissue (Liesman et al., 1995). Again, we found no effect of phlorizin on plasma concentration of GH (Table 2). Finally, we considered the work of Brockman (1984), who demonstrated that hypoglycemia directly stimulated lipolysis in an elegant study of alloxan diabetic, adrenal-denervated sheep. However, phlorizin treatment did not cause hypoglycemia in our current study; in fact, phlorizin did not significantly alter plasma glucose concentration (Table 2). Although we are unable to address the possibility that phlorizin treatment caused a stress response in these animals, Brockman’s observation that phlorizin causes lipolysis in animals without active adrenal glands (1984) demonstrates that a stress response is not a required component of phlorizin-induced lipolysis. Therefore, the cause of phlorizin-induced lipolysis in lactating dairy cows remains unclear. It is possible that small, statistically undetectable changes in plasma insulin and glucose concentration were adequate to increase plasma NEFA concentration to the extent that was observed in this experiment. In addition, despite the validation of plasma NEFA concentration as an index of lipolysis (Dunshea et al., 1989), it is important to note that we did not directly measure lipolytic rate in this experiment. We found a significant stage of lactation x treatment interaction for plasma BHBA concentration (P 0.03), but the reason for this response is unclear.
Phlorizin did not alter yield of milk, milk lactose, milk fat (Table 3), or 3.5% FCM (not shown). However, there was a tendency for an interaction between stage of lactation and treatment for milk protein yield (P 0.15). We also used the milk FA profile to help assess metabolic changes in response to phlorizin injection. Consistent with observed effects on plasma NEFA concentration, long-chain milk FA yield tended to increase with phlorizin treatment (P = 0.07, Table 3). Long-chain FA in milk are derived from circulating FA rather than de novo synthesis in the mammary gland (Barber et al., 1997), so a tendency for increased secretion of long-chain FA suggests that more circulating FA were available for uptake by the mammary gland. We also found that early-lactation cows had a significantly greater proportion of unsaturated FA in milk fat than did late-lactation cows (39.6 vs. 31.3 g/100 g of FA, P 0.001). Greater 9-desaturase activity in early lactation cows (9-desaturase index: 0.40 vs. 0.30, P 0.01) accounted for the majority of the difference in milk FA saturation, in agreement with the findings of Kay et al. (2005). Some proportion of this increase likely reflects 9-desaturase activity in adipose tissue prior to lipolysis, but increased 9-desaturase activity in the mammary gland may have contributed as well.
Contrary to our primary hypothesis, there was no interaction of treatment and stage of lactation for DMI in this study (P = 0.37). This is perhaps not surprising, given that indicators of lipolysis (plasma NEFA concentration and long-chain FA secretion) responded to treatment in a similar manner in both early- and late-lactation cows. However, the results of this study do contradict our original hypothesis that phlorizin would increase DMI by increasing meal size. Phlorizin treatment tended to decrease mean meal size (P = 0.07; Figure 1), which resulted in a compensatory decrease in mean intermeal interval (P = 0.02) and no effect on DMI (P = 0.15; Table 2).
Although we previously showed that phlorizin administration resulted in increased gluconeogenic capacity, we also observed an increase in NEFA available for hepatic oxidation (Bradford and Allen, 2005). In the current study, we again showed that phlorizin increased circulating NEFA, and the total amount of substrate available for oxidation in the liver may have increased with phlorizin treatment. Furthermore, we previously suggested that propionate and acetyl-CoA may interact to limit meal size in cows mobilizing body fat (Oba and Allen, 2003). Cows in negative energy balance often combine high rates of acetyl-CoA production (from FA oxidation) with a relative shortage of glucogenic substrate. This situation results in a deficit of TCA cycle intermediates and a more reduced state of NAD (lower NAD+/NADH) preventing oxidation of acetyl-CoA (Lopes-Cardozo et al., 1975). Propionate can stimulate acetyl-CoA oxidation both by providing TCA intermediates and by utilizing NADH during its conversion to glucose. Such an interaction would lead to rapid ATP production during meals, and could have played a role in the tendency for a decrease in meal size for the phlorizin treatment.
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CONCLUSIONS
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In agreement with past results, phlorizin not only increased glucose demand but also stimulated lipolysis in both early- and late-lactation cows. Phlorizin did not significantly alter plasma concentrations of glucose, insulin, glucagon, or GH. Finally, phlorizin tended to decrease meal size but also decreased intermeal interval, resulting in no change in DMI in either early- or late-lactation cows. Phlorizin provides a model for increased glucose demand, but confounding effects on lipid metabolism complicate interpretation of its effects on hepatic energy metabolism in lactating cows.
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ACKNOWLEDGEMENTS
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The authors thank R. E. Kreft, R. A. Longuski, D. G. Main, Y. Ying, C. S. Mooney, and J. A. Voelker Linton for their technical assistance and West Central Soy for donation of the SoyPlus protein supplement.
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FOOTNOTES
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1 This material is based on work supported by the Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture, under Agreement No. 2004-35206-14167, and under a National Science Foundation Graduate Research Fellowship. 
2 Current address: 127 Call Hall, Kansas State University, Manhattan, KS 66506.
Received for publication September 27, 2006.
Accepted for publication November 27, 2006.
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ABSTRACT
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