K.J. Harvatine

Recent advances in the regulation of milk fat synthesis

In addition to its economic value, milk fat is responsible for many of milks characteristics and can be markedly affected by diet. Diet-induced milk fat depression (MFD) was first described over a century ago and remains a common problem observed under both intensive and extensive management. The biohydrogenation theory established that MFD is caused by an inhibition of mammary synthesis of milk fat by specific fatty acids (FA) produced as intermediates in ruminal biohydrogenation. During MFD, lipogenic capacity and transcription of key lipid synthesis genes in the mammary gland are down-regulated in a coordinated manner. Our investigations have established that expressions of sterol response element-binding protein 1 (SREBP1) and SREBP-activation proteins are down-regulated during MFD. Importantly, key lipogenic enzymes are transcriptionally regulated via SREBP1. Collectively, these results provide strong evidence for SREBP1 as a central signaling pathway in the regulation of mammary FA synthesis. Spot 14 is also down-regulated during MFD, consistent with a lipogenic role for this novel nuclear protein. In addition, SREBP1c and Spot 14 knock-out mice exhibit reduced milk fat similar to the magnitude and pattern of MFD in the cow. Application of molecular biology approaches has provided the latest chapter in the regulation of milk fat synthesis and is reviewed along with a brief background in nutritional regulation of milk fat synthesis in ruminants.

Nutrigenomics, Rumen-Derived Bioactive Fatty Acids, and the Regulation of Milk Fat Synthesis

Mammary synthesis of milk fat continues to be an active research area, with significant advances in the regulation of lipid synthesis by bioactive fatty acids (FAs). The biohydrogenation theory established that diet-induced milk fat depression (MFD) in the dairy cow is caused by an inhibition of mammary synthesis of milk fat by specific FAs produced during ruminal biohydrogenation. The first such FA shown to affect milk fat synthesis was trans-10, cis-12 conjugated linoleic acid, and its effects have been well characterized, including dose-response relationships. During MFD, lipogenic capacity and transcription of key mammary lipogenic genes are coordinately down-regulated. Results provide strong evidence for sterol response element-binding protein-1 (SREBP1) and Spot 14 as biohydrogenation intermediate responsive lipogenic signaling pathway for ruminants and rodents. The study of MFD and its regulation by specific rumen-derived bioactive FAs represents a successful example of nutrigenomics in present-day animal nutrition research and offers several potential applications in animal agriculture.

Expression of Enzymes and Key Regulators of Lipid Synthesis Is Upregulated in Adipose Tissue during CLA-Induced Milk Fat Depression in Dairy Cows

Milk fat depression (MFD) is a naturally occurring condition in dairy cows where milk fat synthesis is inhibited by intermediates of ruminal biohydrogenation. One of these bioactive fatty acids (FA), trans-10, cis-12 conjugated linoleic acid (CLA), decreases milk fat synthesis through transcriptional downregulation of genes involved in mammary lipid synthesis. Energy partitioning during MFD is not well characterized because of the complexity of observing energy metabolism in ruminant animals. To investigate energy partitioning during MFD, adipose tissue biopsies were taken from 4 cows arranged in a switchback design. Treatments were control and 4-d abomasal infusion of trans-10, cis-12 CLA (7.5 g/d). CLA decreased milk fat yield by 38% and milk fat content by 34%, but yields of milk and other milk components were unchanged. In contrast to reported changes in mammary tissue, adipose tissue expression of lipid synthesis enzymes, including lipoprotein lipase, FA synthase, stearoyl-CoA desaturase, and FA binding protein 4, was increased. Expression of regulators of lipid synthesis, including sterol-response element binding protein 1, thyroid hormone responsive spot 14, and PPARgamma, also increased in adipose tissue. Thus, a CLA dose resulting in near maximal inhibition of mammary lipid synthesis resulted in increased expression of lipid synthesis-related genes in adipose tissue. A meta-analysis of intake response during CLA infusion was conducted to extend the investigation of energy metabolism during MFD. Voluntary intake decreased (P < 0.001) by 1.5 kg/d during CLA-induced MFD in the 14 studies analyzed, but the reduction in intake only partially accounts for the energy spared from reduced milk fat synthesis. Results are consistent with energy spared from the reduction in milk fat synthesis being partitioned toward adipose tissue fat stores during short-term MFD.

Induction of and recovery from milk fat depression occurs progressively in dairy cows switched between diets that differ in fiber and oil concentration

Milk fat depression (MFD) caused by intermediates of ruminal biohydrogenation commonly occurs in dairy cattle. The time course of recovery from MFD is important to mechanistic investigation and management of the condition. Nine cows were used in a repeated design, allowing analysis of recovery from diet-induced MFD. A high-fiber, low-oil diet was fed during the control and recovery periods, and a low-fiber, high-oil (LFHO) diet was fed during the induction period. Milk yield was not affected by treatment. Milk fat percentage and yield decreased progressively during induction and were lower by d 3 and 5, respectively. Milk fat concentration and yield increased progressively when cows were fed the recovery diet and were not different from control on d 19 and 15, respectively. Yield of de novo synthesized fatty acids (FA) decreased progressively during the induction period and was lower than that of controls by d 5. A biphasic response was seen for milk fat trans isomers, where trans-11 C18:1 and cis-9,trans-11 conjugated linoleic acid (CLA) were elevated initially and trans-10 C18:1 and trans-10,cis-12 CLA increased progressively during the induction period. A similar biphasic response was seen during recovery from MFD, with trans-10 C18:1 and trans-10,cis-12 rapidly decreasing initially and trans-11 C18:1 and cis-9,trans-11 CLA increasing slightly above control levels during the second phase. Recovery from diet-induced MFD occurs gradually with a short lag when dietary fiber and oil concentrations are corrected. This time course provides a framework to identify factors causing MFD and set expectations during recovery from MFD.

Plasma FGF21 Is Elevated by the Intense Lipid Mobilization of Lactation

In many mammals, lactation success depends on substantial use of lipid reserves and requires integrated metabolic activities between white adipose tissue (WAT) and liver. Mechanisms responsible for this integration in lactation are poorly understood, but data collected in other conditions of elevated lipid use suggest a role for fibroblast growth factor-21 (FGF21). To address this possibility in the context of lactation, we studied high-yielding dairy cows during the transition from late pregnancy (LP) to early lactation (EL). Plasma FGF21 was nearly undetectable in LP, peaked on the day of parturition, and then stabilized at lower, chronically elevated concentrations during the energy deficit of EL. Plasma FGF21 was similarly increased in the absence of parturition when an energy-deficit state was induced by feed restricting late-lactating dairy cows, implicating energy insufficiency as a cause of chronically elevated FGF21 in EL. Gene expression studies showed that liver was a major source of plasma FGF21 in EL with little or no contribution by WAT, skeletal muscle, and mammary gland. Meaningful expression of the FGF21 coreceptor β-Klotho was restricted to liver and WAT in a survey of 15 tissues that included the mammary gland. Expression of β-Klotho and its subset of interacting FGF receptors was modestly affected by the transition from LP to EL in liver but not in WAT. Overall, these data suggest a model whereby liver-derived FGF21 regulates the use of lipid reserves during lactation via focal actions on liver and WAT.

Dietary unsaturated fatty acids increase plasma glucagon-like peptide-1 and cholecystokinin and may decrease premeal ghrelin in lactating dairy cows.

Previous reports have indicated that dietary unsaturated fat can decrease energy intake of lactating dairy cattle. However, the mechanism for this response is unclear. To evaluate the potential role of gut peptides, periprandial concentrations of cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1), and ghrelin were measured. From a replicated 4 x 4 Latin square experiment, 4 cows from a single square were selected for analysis of responses to 3 treatments: a control diet (5.5% total fatty acids, 65% unsaturated), a diet with added saturated fat (SAT, 8.3% fatty acids, 47% unsaturated), and a diet with added unsaturated fat (UNS, 7.8% fatty acids, 63% unsaturated). The SAT treatment increased duodenal flow of saturated fatty acids compared with UNS and control and, despite the fact that ruminal biohydrogenation altered fatty acid profiles of digesta, UNS increased duodenal flow of unsaturated fatty acids compared with SAT and control. Blood samples were collected at 8-min intervals through the first 2 meals of the day and analyzed by commercial radioimmunoassays. The UNS treatment increased plasma CCK concentration relative to SAT and control, and increased plasma GLP-1 concentration compared with control. Furthermore, fat treatments tended to suppress the prandial ghrelin surge that was evident for control. Suppression of feed intake by unsaturated fats is likely mediated in part by increased secretion of CCK and GLP-1, and dietary fat may also inhibit ghrelin release before conditioned meals.

Characterization of the acute lactational response to trans-10, cis-12 conjugated linoleic acid

Trans-10, cis-12 conjugated linoleic acid (CLA) is a potent inhibitor of milk fat synthesis in the dairy cow. The decrease in milk fat yield during abomasal infusion of CLA reaches a nadir after 3 to 5 d. The acute responses to CLA were evaluated using 4 cows in a crossover design. Cows were milked with the aid of oxytocin every 4h from -28 to 80h and every 6h from 86 to 116h relative to the initiation of abomasal CLA infusion. An initial priming dose of 7.5g of CLA was given at time zero followed by infusion of 2.5g every 4h for 72h. Plasma CLA reached a near-steady-state concentration by 4h, and initial plasma enrichments were greatest in the triglyceride and nonesterified fatty acid fractions. Milk CLA concentration peaked at 6h and reached steady state by 22h. At termination of the infusion, decreases in milk CLA concentration and yield and plasma CLA concentration were best fit by a reciprocal-linear function. Milk fat percentage decreased progressively after 2h and was significant by 14h. Milk fatty acid profile was initially unchanged, but between 18 and 36h after initiation of the CLA dose the proportions of fatty acids progressively shifted, resulting in an increase in fatty acids >C16 and a decrease in fatty acids <C16 by 38 to 46h. In contrast, changes in the desaturase index were immediate, with a significant decrease by 6h and a near-maximal decrease by 10h. Thus, stearoyl desaturase enzyme was more acutely responsive to CLA than other enzymes in milk fat synthesis. The initial decrease in milk fat synthesis involved an equal depression of short- and long-chain fatty acid pathways and was followed thereafter by a more pronounced decrease in the synthesis of de novo fatty acids.

Dietary cocoa reduces metabolic endotoxemia and adipose tissue inflammation in high-fat fed mice

In diet-induced obesity, adipose tissue (AT) is in a chronic state of inflammation predisposing the development of metabolic syndrome. Cocoa (Theobroma cacao) is a polyphenol-rich food with putative anti-inflammatory activities. Here, we examined the impact and underlying mechanisms of action of cocoa on AT inflammation in high fat-fed mice. In the present study, male C57BL/6 J mice were fed a high fat diet (HF), a HF diet with 8% (w/w) unsweetened cocoa powder (HFC), or a low-fat diet (LF) for 18 weeks. Cocoa supplementation decreased AT mRNA levels of tumor necrosis factor-α, interleukin-6, inducible nitric oxide synthase, and EGF-like module-containing mucin-like hormone receptor-like 1 by 40-60% compared to HF group, and this was accompanied by decreased nuclear protein levels of nuclear factor-κB. Cocoa treatment reduced the levels of arachidonic acid in the AT by 33% compared to HF controls. Moreover, cocoa treatment also reduced protein levels of the eicosanoid-generating enzymes, adipose-specific phospholipase A2 and cyclooxygenase-2 by 53% and 55%, respectively, compared to HF-fed mice. Finally, cocoa treatment ameliorated metabolic endotoxemia (40% reduction in plasma endotoxin) and improved gut barrier function (as measured by increased plasma levels of glucagon-like peptide-2). In conclusion, the present study has shown for the first time that long-term cocoa supplementation can reduce AT inflammation in part by modulating eicosanoid metabolism and metabolic endotoxemia.

Effect of a high-palmitic acid fat supplement on milk production and apparent total-tract digestibility in high- and low-milk yield dairy cows

The effect of a high-palmitic acid fat supplement was tested in 12 high-producing (mean = 42.1 kg/d) and 12 low-producing (mean = 28.9 kg/d) cows arranged in a replicated 3 × 3 Latin square design. Experimental periods were 21 d, with 18d of diet adaptation and 3 d of sample collection. Treatments were (1) control (no supplemental fat), (2) high-palmitic acid (PA) supplement (84% C16:0), and (3) Ca salts of palm fatty acid (FA) supplement (Ca-FA). The PA supplement had no effect on milk production, but decreased dry matter intake by 7 and 9% relative to the control in high- and low-producing cows, respectively, and increased feed efficiency by 8.5% in high-producing cows compared with the control. Milk fat concentration and yield were not affected by PA relative to the control in high- or low-producing cows, although PA increased the yield of milk 16-C FA by more than 85 g/d relative to the control. The Ca-FA decreased milk fat concentration compared with PA in high-, but not in low-producing cows. In agreement, Ca-FA dramatically increased milk fat concentration of trans-10 C18:1 and trans-10, cis-12 conjugated linoleic acid (>300%) compared with PA in high-producing cows, but not in low-producing cows. No effect of treatment on milk protein concentration or yield was detected. The PA supplement also increased 16-C FA apparent digestibility by over 10% and increased total FA digestibility compared with the control in high- and low-producing cows. During short-term feeding, palmitic acid supplementation did not increase milk or milk fat yield; however, it was efficiently absorbed, increased feed efficiency, and increased milk 16-C FA yield, while minimizing alterations in ruminal biohydrogenation commonly observed for other unsaturated fat supplements. Longer-term experiments will be necessary to determine the effects on energy balance and changes in body reserves.

Lipid feeding and milk fat depression.

Diets fed to cattle contain mostly unsaturated fatty acids supplied in grains and forages, by-products, and fat supplements. Lipid intake by dairy cattle must be restricted to prevent alterations of microbial populations in the rumen that can negatively affect milk yield. Unsaturated fatty acids consumed by cattle are extensively metabolized through biohydrogenation, intermediates of which include conjugated linoleic acid (CLA) and trans-monoenoic acid isomers. Three specific CLA intermediates of biohydrogenation have been shown to cause milk fat depression in dairy cattle through coordinated suppression of mammary lipogenic genes by a transcription factor that is a central regulator of lipid synthesis.