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Ross Products Division Abbott Laboratories, Columbus, OH and * Metabolex, Hayward, CA
2To whom correspondence should be addressed. E-mail: neile.edens{at}rossnutrition.com.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • diabetes • phytochemicals • plant extract
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Over thousands of years, the human body has evolved in the presence of a diet quite different from that eaten in the 21st century. As assessed from studies of Paleolithic nutrition and contemporary hunter-gatherer societies, the diet of ancient humans was characterized by foods with a high micronutrient-to-energy ratio (2
), rich in unrefined plant foods. It may be inferred that the human body is adapted to, and operates optimally, when fed such a diet. The standard American diet today is characterized by excess energy, is relatively poor in plant food and is depleted of nutrients by refining. Consumption of this diet may contribute to a proportion of life-style related diseases (3
), including some types of cancer, cardiovascular disease and diabetes mellitus. Epidemiologic evidence suggests that consumption of a diet low in energy, high in plant food and rich in unrefined products might reduce the occurrence of these diseases; high fruit and vegetable intake decreases the risk of developing diabetes mellitus (4
,5
).
The prevalence of both obesity and type 2 diabetes mellitus is increasing in the developed world (6
). Type 2 diabetes is preceded by impaired glucose tolerance, in which there is defective first-phase insulin secretion, insulin resistance and hyperinsulinemia but normoglycemia (7
). As the disease progresses, the pancreatic beta cells fail, resulting in hyperglycemia and associated complications. Type 2 diabetes is diagnosed by elevated levels of fasting serum insulin and glucose, and by abnormally high concentrations of blood glucose after an oral glucose load.
Type 2 diabetes is treated, at least in part, by nutritional intervention. In the West, recommendations typically include maintenance of optimal weight and control of carbohydrate intake. Several plant foods improve glucose tolerance in vivo (defined as the blood glucose excursion after an oral glucose load) (8
–10
, 12
13
). Many of these have been indexed in the NAPRAlert database, at the University of Illinois (11
) including brewer’s yeast (Saccharomyces cerevisiae) (14
). The mechanism of action of these foods is not yet known.
Food components that improve glucose tolerance may act in the gut by inhibiting digestion and/or absorption of carbohydrates (15
), on the pancreas by stimulating insulin secretion (16
) or on insulin-sensitive tissues, such as adipose tissue and muscle, by stimulating glucose uptake (17
) and metabolism (18
). Food components that enhance glucose uptake and metabolism may act as "insulin-mimetic" agents, which work in the absence of insulin, or as "insulin-potentiating agents," which enhance the action of insulin but have no independent effects on glucose metabolism. These processes can be studied in vitro, by using cells or tissue fragments isolated from insulin-sensitive organs.
Brewer’s yeast has long been studied as a source of glucose tolerance factor (GTF),3
a Cr-rich yeast extract. When given orally to rats, GTF repairs the glucose intolerance induced by feeding a Cr-depleted diet (19
). GTF also stimulates glucose utilization by cells in vitro (20
). The active component of GTF has never been identified with certainty, but previously described yeast components implicated in glucose tolerance include a nicotinic acid-glutathione-Cr(III) complex (21
), a quinoline derivative (22
) and phosphatidylinositol glycans (23
). We have developed a novel yeast extract (YE) which is a complex mixture of inorganic and organic species.
The purpose of this work was to explore whether YE, like previously described GTF and other yeast extracts, affects metabolism in insulin-sensitive cells. This was accomplished by determining the effect of YE on rat adipocyte glucose transport, lipid synthesis and lipolysis. The YE was fractionated by reverse-phase chromatography. The activity of the resulting fractions, each containing a mixture of components, was assessed in vitro.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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Bovine serum albumin (BSA; Type V), adenosine deaminase, and glycerokinase were purchased from Boerhinger-Mannheim/Roche Biochemical (Indianapolis, IN). Collagenase (Type I) was purchased from Worthington Biomedical (Lakewood, NJ). Recombinant human insulin (Humulin-R) was purchased from Eli Lilly (Indianapolis, IN). U-14C-glucose was purchased from DuPont NEN Research Products (Boston, MA). Cytoscint scintillation cocktail was purchased from ICN Biomedical (Costa Mesa, CA). Components for media were purchased from Sigma Chemical (St. Louis, MO) and ICN Biomedical. All additional reagents for the glycerol assay, methylenediphosphonic acid trisodium salt, 1,2-diaminocyclohexane tetraacetate and tetrabutyl ammonium hydrogen sulfate for the analytical analysis were purchased from Sigma Chemical. Organic solvents for open column chromatography were purchased from Fisher Scientific (Pittsburgh, PA) and for HPLC from Burdick and Jackson (Muskegon, MI). D2O and NaOD for nuclear magnetic resonance (NMR) analysis were purchased from Isotec (Miamisburg, OH) and Wilmad Glass (Buena, NJ), respectively. For HPLC, mono- and dibasic potassium phosphate were purchased from Mallinckrodt Baker (Paris, KY).
Yeast extract fractionation.
YE is a proprietary extract of brewer’s yeast. The yield of YE is 2.9% of the starting material. YE was further fractionated by reverse phase chromatography on a open C18 column (Supelco Supelclean ENVI-18, Bellefonte, PA) with 0.48 mol/L ammonium acetate buffer at pH 5.8 with a 40% acetonitrile, in ammonium acetate buffer, elution solvent. The fractionation process was monitored by conductivity and UV absorbance at 280 nm. Fraction 1 (Fx1) eluted unretained from the column at relatively high conductivity. Fraction 2 (Fx2) was retained only slightly on the column and like Fx1, eluted with ammonium acetate buffer. Fraction 3 (Fx3) was eluted from the column with acetonitrile (40%) in ammonium acetate.
Analysis of YE.
Mineral content of YE was determined by microwave digestion in nitric acid, and subsequent analysis using an ARL Accuris ICP-AES (Thermo Elemental, Franklin, MA). Chromium and molybdenum were measured using an ultrasonic nebulizer (Cetac U-500 AT, Omaha, NE) to enhance sensitivity. All other minerals were analyzed using a standard Babington nebulizer (Cetac). Quantitation was by comparison with NIST-traceable standards. Chloride was determined by potentiometric titration with silver nitrate (24
).
Fat was determined by the Rose-Gottlieb method of solvent extractable total solids (25
). Moisture was determined as loss on drying using a vacuum oven (26
). Total nitrogen was determined by the Kjeldahl method using a Tecator Kjeltec System (Foss North America, Eden Prairie, MN) (27
). Carbohydrate was determined as total hexose by standard methods (28
). Nucleotides were analyzed as described and reported as the sum of UMP, CMP, IMP, GMP and AMP (29
).
Amino acids were determined after acid hydrolysis by ion exchange separation with postcolumn ninhydrin derivitization using the Beckman System 6300 Amino Acid Analyzer (Beckman Instruments, Fullerton, CA). Cyst(e)ine was determined (using the same instrumentation) after complete conversion to cysteic acid as described (30
). The other amino acids reported were determined by standard methods (31
).
An ion-pair reversed phase HPLC method was developed for fingerprinting of YE and the fractions generated with an open C18 column. The chromatographic profiles of all YE samples were performed in 157 min on a 5-µm Supercosil LC-18-T reversed phase analytical column (250 x 4.6 mm i.d.; Supelco) combined with a suitable guard column at room temperature. The chromatography was carried out using a Waters system pump 616 (Milford, MA) with a manual injector, loop 50 µL. The absorbance was monitored at 214 nm and 254 nm with the range of sensitivity 0.01 (Spectra-Physics detector SP8490, Mountain View, CA). The mobile phase contained tetrabutylammonium hydrogen sulfate as an ion-pairing agent and potassium phosphate buffer to improve the separation of ionic components of the YE and fractions (Table 1
). Mobile phases C and D were applied for strongly binding analytes that were difficult to elute without lower levels of buffer, ion-pairing agent and higher concentrations of organic solvent. These phases yielded broad nondescriptive profiles in the region between 90 and 157 min, which are not included in this discussion.
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) reference at 16.84 ppm (referenced against 85% phosphoric acid, = 0.0 ppm) and 1,2-diaminocyclohexane tetraacetate was added at an approximate final concentration of 20 mmol/L as a chelator of paramagnetic cations. Final pH was adjusted with NaOD. The 31P NMR spectra were collected using a standard pulse sequence and WALTZ proton-decoupling scheme on a Varian Unity 500 MHz spectrometer (Palo Alto, CA) equipped with a 3-mm Nalorac (Martinez, CA) dual tunable probe (13C and 31P). All spectra were collected at an equilibrated temperature of 27°C. Animals.
Young male Sprague-Dawley rats (130–170 g) from Harlan (Indianapolis, IN) were housed 3–4 per plastic tub with sawdust chips. Lights were on at 0600 h, off at 1800 h. The rats consumed Harlan Teklad nonpurified diet and water ad libitum. The rats were acclimated to the laboratory for at least 6 d, then killed by decapitation in groups of 3 or 4 and their epididymal and retroperitoneal fat pads dissected and pooled for adipocyte isolation. The protocol for animal use was approved by the Institutional Laboratory Animal Care and Use Committee of the Ohio State University.
Adipocyte isolation.
The adipocytes were isolated from the pooled fat pads by digestion using a collagenase solution prepared in Krebs-Ringer’s-HEPES with 25 g/L BSA (KRH-BSA); (32
). In digests to be used for glucose transport measurements, the solution contained 0.5 mmol/L glucose, whereas in digests to be used for glucose metabolism/lipolysis, it contained 5 mmol/L glucose.
Glucose transport.
Glucose transport was measured by standard methods with minor modifications (33
). Isolated adipocytes were preincubated for 1 h in KRH-BSA lacking glucose in vitro with insulin (51 pmol/L), YE (10 mg/L) or insulin plus YE before transport of U-14C-glucose [3.85 µmol/L, specific activity (SA) = 577 x 106 dpm/µmol] was measured for 45 min. Adipocytes were separated from medium by centrifugation (16,000 x g, 2 min) through silicon oil. Transport was assessed by uptake of 14C-glucose into adipocytes by counting in a liquid scintillation counter (Packard TriCarb 2500TR, Downer’s Grove, IL). Triplicate measurements were made for each variable in each experiment. All values were corrected for zero time transport.
Glucose metabolism and lipolysis.
Glucose metabolism and lipolysis were determined simultaneously (34
). Isolated adipocytes were incubated in medium (KRH-BSA) with glucose (5 mmol/L), sodium bicarbonate (10 mmol/L), palmitic acid (0.25 mmol/L), and adenosine deaminase (800 U/L) with insulin (60 pmol/L), YE (10 mg/L) or insulin plus YE. After 1 h incubation, medium was separated from adipocytes by gentle centrifugation (1000 x g, 1 min) and collected for subsequent analysis. Lipids were extracted from adipocytes with isopropanol/heptane/H2SO4 (1 mol/L), 40:10:1. Glucose metabolism was measured by incorporation of U-14C-glucose (SA = 0.89 x 106 dpm/µmol) into total lipids. Lipolysis was measured by assay of glycerol release into the medium. Medium was separated from cells by gentle centrifugation and collected by aspiration. Medium samples were stored at -80°C until glycerol assay. Triplicate measurements were made for each variable in each experiment. All values were corrected for zero time incubation.
Glycerol determination.
The concentration of glycerol in the medium was measured using an enzymatic, fluorimetric method (35
) modified to a 96-well plate format (Cytofluor4000, Applied Biosystems, Foster City, CA). Each sample was assayed in duplicate.
Calculations and statistics.
To determine whether YE acted as an insulin-mimetic or as an insulin potentiator, we performed the following calculations on glucose metabolism data (dpm 14C-glucose incorporated into lipid):
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Data were analyzed by two-way repeated-measures ANOVA (Systat 6.0.1, SPSS, Chicago, IL) followed by post-hoc Bonferroni t tests to compare pairs of interest. Differences were considered significant if P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Large amounts of salt were present; the bulk of this was added during processing.
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In preliminary experiments, a range of YE concentrations was tested (0.1–1000 mg/L). Glucose metabolism to lipid was responsive to the concentration of YE (data not shown).The lowest reliably active concentration was 10 mg/L, which was chosen therefore for further investigation. Insulin (60 pmol/L) stimulated glucose metabolism to lipid in rat adipocytes 160% (Fig. 1
, P < 0.001 vs. basal). YE (10 mg/L) stimulated glucose metabolism to lipid 14% (P = 0.001). The combination of YE and insulin was more potent than insulin alone (P = 0.006), i.e., YE potentiated the effect of insulin on glucose metabolism by 30%.
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Preliminary experiments were performed to assess the insulin dose-response curve for glucose transport. The concentration of insulin that stimulated half-maximal glucose transport was found to be 51 pmol/L (Fig. 2
, inset). This concentration of insulin was used in subsequent glucose transport experiments. Although insulin (51 pmol/L) stimulated glucose transport to sevenfold the basal value (P = 0.02), YE (10 mg/L) did not affect glucose transport, nor did YE affect insulin stimulation of glucose transport (Fig. 2)
.
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Preliminary experiments showed that YE alone inhibited lipolysis linearly from 1 to 100 mg/L. YE (100 mg/L) inhibited lipolysis by 91% (data not shown). Insulin (60 pmol/L), YE (10 mg/L) and the combination inhibited lipolysis by 46, 62 and 81%, respectively (Fig. 3
; P < 0.001). The effects of YE and insulin were partially additive.
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The fractionation of YE (40.6 g) gave three fractions, i.e., Fx1, 14.3 g; Fx2, 10.39 g; and Fx3, 9.7 g. Hydrophobicity increased and polarity decreased with increasing fraction number. Recovery was 85%. Ion-pairing HPLC analysis of unfractionated YE and Fx1–3 revealed that the fractions were still extremely complex, containing many compounds that absorb at 214 nm (Fig. 4
) and 254 nm (not shown).
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).
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As seen before, insulin stimulated glucose metabolism compared with no additions by 164% (P = 0.002), (Fig. 6
). Unfractionated YE and Fx3 alone stimulated glucose metabolism over basal by 27 and 41%, respectively (P < 0.05). In this experiment, YE plus insulin did not significantly increase glucose metabolism to lipid compared with insulin alone; however, Fx2 plus insulin stimulated glucose metabolism to lipid by 25% compared with insulin alone (P < 0.05). The potentiating effect of Fx3 on insulin stimulation (35%) was not significant (P = 0.067).
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. Although YE and Fx1 were inactive, both Fx2 (P = 0.034) and Fx3 (P = 0.130) potentiated insulin stimulation of glucose metabolism to lipid (Fig. 7
).
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To determine whether fractionation concentrated the antilipolytic activity of YE, the same fractions were tested for their ability to enhance the antilipolytic effect of insulin. YE (10 mg/L) and all its fractions decreased lipolysis in the presence of insulin (60 pmol/L), but to markedly different degrees (Fig. 8
). Antilipolytic activity was retained, if not concentrated, in Fx2 (74%, P = 0.001) and Fx3 (69%, P < 0.001), whereas Fx1 inhibited lipolysis by only 12% (P = 0.277).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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YE mimicked or enhanced the action of insulin, but the nature and magnitude of its effect depended on the pathway under study. YE mimicked the action of insulin on lipolysis in rat adipocytes in vitro. In contrast, YE did not affect glucose transport into adipocytes either alone or in the presence of insulin. Despite this lack of effect on glucose transport, YE modestly stimulated glucose incorporation into lipid. YE may enhance glucose metabolism to lipid without stimulating glucose transport by altering the distribution of glucose among various metabolic pathways in fat cells. We hypothesize that YE may decrease glucose metabolism to lactate by adipocytes (36
). Calculations revealed that YE potentiates the action of insulin on glucose metabolism more than can be accounted for by the insulin-mimetic effect of YE alone. In contrast, the combined antilipolytic effects of YE and insulin are less than additive. We hypothesize that the apparent lack of additivity is attributable to the potency of YE on this pathway because lipolysis may have been nearly maximally inhibited by the concentration of YE used. Use of lower YE doses may reveal a potentiating effect of YE on the antilipolytic effect of insulin.
GTF stimulates glucose oxidation in yeast and adipocytes (20
,37
). Similarly, other investigators have found that a yeast extract stimulates glucose transport into adipocytes in the absence of insulin, but very little in the presence of insulin (38
). The reason for the discrepancy between those results and the present findings is unknown but may be attributable to methodological differences in the preparation of the extract or the transport assay. Similar to our results, other investigators have found that GTF enhances the action of insulin on glucose metabolism to lipid in adipocytes (20
,39
). There are no previous reports that GTF or other yeast extracts inhibit lipolysis in adipocytes.
The mechanism of action of YE in vitro is unknown. Because YE does not affect glucose transport, works as an insulin potentiatior on glucose metabolism to lipid and is an insulin mimetic on lipolysis, it is likely that YE works on multiple targets in the adipocyte. Alternatively, YE may act on a process or pathway common to insulin-stimulated lipid synthesis and antilipolysis. YE may potentiate insulin activation of acetyl CoA carboxylase, the rate-limiting step in fatty acid synthesis, and also stimulate phosphodiesterase 3B, and consequently, the rate of lipolysis. Elucidation of the signaling pathway mediating the effects of YE awaits identification of its active component(s).
The active component(s) in YE is (are) not yet known. Compositional analysis, ion-pairing HPLC and 31P NMR provided a more complete picture of the components of YE, and fractions prepared from it, than was available previously. The HPLC-UV data suggest that the mixtures are enriched in purine- and pyrimidine-containing compounds based on similar profiles at 214 and 254 
(40
). The 31P NMR data contain resonances consistent with a complex mixture of phosphorylated biochemicals, including phosphomonoesters, phosphodiesters and polyphosphates (41
); however, additional experimentation is required for specific compound identification. Examples of these classes of compounds, which have been previously reported as components of yeast extracts (42
), are phosphorylated monosaccharides and mononucleotides, glycerol phosphodiesters and nucleoside triphosphates, respectively. The 31P NMR data also suggest that YE contains inorganic phosphate.
To reduce this complexity, YE was fractionated by open column chromatography to yield three fractions that differ in hydrophobicity, polarity and inorganic content. These three fractions are roughly equal in mass, but may be expected to have differing composition. Fx1, obtained from column flow-through, is relatively enriched in minerals, salt, inorganic phosphate and hydrophilic organic compounds, whereas Fx3, eluted with 40% acetonitrile, is more enriched in organic compounds that are more hydrophobic and less polar than those in Fx1. Fx2, eluted with ammonium acetate buffer, contains components found in both Fx1 and Fx3. Components common to Fx2 and Fx3 include complex nucleotides and peptides. Fx1 (least hydrophobic) was relatively inactive in glucose metabolism and lipolysis assays. Fx2 and Fx3 (more hydrophobic) were relatively more potent in these assays, suggesting that relatively nonpolar molecules are responsible for the insulin-mimetic and insulin-potentiating effects of YE.
Previous investigators attempted to identify the active component of GTF by fractionations guided by in vitro assays (yeast, adipocyte or hepatocyte). It has been suggested that amino acids and nucleosides may be involved in the insulin-potentiating effect of yeast extract (43
), whereas others have emphasized the role of Cr(III) (21
), especially as complexed with nicotinic acid and glutathione. YE is comparatively low in Cr; analysis revealed 5 µg/g (final concentration in vitro was 50 ng/L) compared with levels of 10–20 µg Cr/g yeast extract (21
). The role of Cr(III) may have been exaggerated by testing yeast extracts on Cr-deficient yeast or adipocytes derived from rats fed a torula yeast diet, which are uniquely responsive to the insulin-potentiating effects of Cr. More recent investigators have found other biologically active components in yeast. Adenosine has been proposed as an active component in yeast extract (43
), but cannot be responsible for the effects we have observed because we included the enzyme adenosine deaminase to convert adenosine to inosine and hypoxanthine (44
). A quinoline derivative (22
) and phosphatidylinositol glycan (23
) have been identified as insulin-mimetic components of yeast extract. It is not known whether the YE used in the present experiments contains these compounds.
Our results, obtained with ion-pairing HPLC and 31P NMR methods, indicate that YE is more complex than previously known. We have extended the insulin-mimetic actions of YE to include antilipolysis, in addition to previously described effects on glucose uptake and metabolism. The potent activity of YE in vitro suggests that it may decrease blood glucose in vivo. Further work must be done to ascertain the bioavailability and activity of YE fractions and components in animal models of diabetes mellitus.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BSA, bovine serum albumin; Fx1, fraction 1; Fx2, fraction 2; Fx3, fraction 3; GTF, glucose tolerance factor; KRH-BSA, Krebs-Ringer’s-HEPES with 25 g/L BSA; NMR, nuclear magnetic resonance; SA, specific activity; YE, yeast extract.
Manuscript received 5 November 2001. Initial review completed 4 January 2002. Revision accepted 11 March 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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