Alcohol is a widely consumed, nonessential, bioactive nutrient with end-organ effects on the skeleton. Moderate levels of alcohol consumption are generally associated with increased bone mineral density (BMD), whereas higher intake levels result in reduced bone formation, bone loss, and increased risk for osteoporotic fracture. Given the negative impact of poor bone health on quality of life and mortality, as well as the knowledge that the majority of adults in America consume alcohol, it is critical to improve our understanding of how alcohol exerts its effects on the human skeleton. The precise mechanism(s) mediating the beneficial and detrimental actions of alcohol on bone are uncertain. Alcohol and its metabolites have direct, as well as indirect, effects on the skeleton. Studies performed in humans, rodents, and nonhuman primates suggest that alcohol-induced reductions in insulin-like growth factor 1 (IGF-1) contribute to the effects of alcohol on bone metabolism. IGF-1 is an important bone anabolic agent that is produced in the liver (and released into circulation) and locally in the skeleton. The central hypothesis of this dissertation was that (1) ethanol consumption is associated with a dose-dependent reduction in bone turnover, (2) the reduced bone formation in alcohol-consuming monkeys is closely associated with decreased systemic and/or skeletally produced IGF-1, and (3) alcohol withdrawal results in parallel increases in serum IGF-1 and osteocalcin, suggesting that abstinence reverses the inhibitory effect of alcohol on bone formation. To test this hypothesis, I capitalized on a unique opportunity to evaluate tissue collected from late adolescent and young adult cynomolgus and rhesus macaques as part of an ongoing alcohol research project sponsored by the National Institute of Alcohol Abuse and Alcoholism (NIAAA) and conducted at the Oregon National Primate Research Center (OHSU; Kathy Grant, PI).
Chapter 1 comprises a literature review which provides an overview of 1) the non-human primate model of voluntary ethanol consumption that is utilized for the present dissertation, including study design and the multi-disciplinary research findings resulting from the model; 2) bone physiology, particularly concerning key concepts investigated in this dissertation including bone remodeling and factors which may influence bone turnover balance; and 3) the skeletal effects of ethanol consumption, with an emphasis on the effects of ethanol on bone turnover and possible mechanisms governing these effects.
The research within this dissertation relied on the measurement of osteocalcin and carboxyterminal cross-linking telopeptide of type 1 collagen (CTX), biochemical markers of bone formation and resorption, respectively, to assess bone turnover. These markers are subject to considerable biological variation in humans, but this has not been closely evaluated in late adolescent or young adult macaques. Chapter 2 presents the results of a study investigating (1) age, sex, and species differences in osteocalcin, CTX, and the ratio of osteocalcin to CTX and (2) within-subject and between-subject variation in biomarkers of bone turnover in late adolescent and young adult rhesus and cynomolgus macaques who had not been habituated to ethanol consumption. Subgroup analyses revealed sex differences for osteocalcin but not CTX among rhesus macaques aged 3.7-6.1 years and cynomolgus macaques aged 6.0-7.0 years, with higher osteocalcin levels in males than females for both species. However, similar patterns emerged between cynomolgus and rhesus macaques, which suggests that there is no advantage or disadvantage in using either of these two species. The study also determined that there is considerable within-subject variation in levels of osteocalcin, CTX, and the ratio of osteocalcin, but there is overall stability of these parameters across subjects over a 4-month interval. The results suggest that while these biomarkers are not ideal for monitoring an individual’s bone turnover over time, they are well-suited for evaluating bone turnover at the population level. Importantly, this supports the use of the biomarkers for assessing the effects of ethanol on bone turnover in the non-human primate model of voluntary ethanol consumption.
After establishing the variation in the levels of biochemical markers of bone turnover, I began to investigate the effects of ethanol on the bone turnover markers. Chapter 3 presents the results of a dose-response study examining the effects of graded increases in ethanol intake on bone turnover in young adult male cynomolgus macaques. Briefly, monkeys consumed 0.5 g/kg/d, 1.0 g/kg/d, and 1.5 g/kg/d ethanol for three consecutive 30-day periods. Over the 90-day interval, there were dosedependent reductions in osteocalcin, CTX, and the ratio of osteocalcin to CTX. These data are indicative of reduced bone formation and resorption, and overall reduced bone turnover, with the reduction of bone formation outpacing the reduction of bone resorption. The study did not detect a threshold for the inhibitory effects of ethanol on bone turnover, suggesting that the effects may occur at low to moderate levels of ethanol consumption. These findings support our hypothesis that ethanol consumption is associated with a dose-dependent reduction in bone turnover. However, the study did not detect a threshold for the inhibitory effects of ethanol on bone turnover, suggesting that the effects may occur at low to moderate levels of ethanol consumption.
I next examined the short-term response of bone turnover markers to ethanol consumption and began to investigate the role of IGF-1 in ethanol-induced alterations in bone turnover in Chapter 4. The study determined that osteocalcin and CTX were suppressed following ethanol consumption, while the ratio of osteocalcin to CTX increased. Levels of the markers were stable prior to ethanol consumption but varied during a 20-hour period following ethanol consumption. In contrast, IGF-1 levels varied during the 20-hour period prior to ethanol consumption and stabilized following ethanol consumption. Additionally, ethanol consumption resulted in a transient increase in insulin-like growth factor 1 binding protein 1 (IGFBP-1) levels. Furthermore, there were correlative relationships between IGF-1 and markers of bone turnover before, but not following, ethanol consumption. While these results do not support our hypothesis that the reduced bone formation in alcohol-consuming monkeys is closely associated with decreased systemic and/or skeletally produced IGF-1, they do suggest that ethanol consumption alters the normal variation of bone turnover markers and IGF-1 in male rhesus macaques, and IGF-1 resistance may play a role in ethanol-induced alterations in bone turnover.
Finally, given the evidence supporting the inhibitory effects of heavy alcohol consumption on bone turnover and the normal relationship between IGF-1 and markers of bone turnover, I sought to determine whether abstinence is sufficient to reverse these effects. Chapter 5 presents a study evaluating the effects of abstinence from ethanol on biochemical markers of bone turnover and IGF-1 in young adult male rhesus macaques. This study determined that abstinence results in decreased osteocalcin, minimal or no change in CTX, and either a reduction or no change in global bone turnover balance depending on the abstinence period. There was little interaction between treatment and time suggesting that, in general, the biochemical markers in the controls parallel the alcohol-treated monkeys during abstinence. Furthermore, the results suggest that short-duration abstinence may be insufficient to restore the normal relationship between IGF-1 and osteocalcin. Additionally, cortisol levels increased during each abstinence period and levels did not differ significantly between treatment and control animals, which suggests that stress may be an important factor to consider when evaluating the effects of abstinence on bone turnover in chronic alcohol users. These results do not support our hypothesis that alcohol withdrawal results in parallel increases in serum IGF-1 and osteocalcin. The similar patterns observed for the bone turnover markers and cortisol between treatment and control animals suggest that stress during involuntary abstinence may exert a greater influence on bone turnover balance than abstaining from alcohol alone.
The central hypothesis of this dissertation was partially accepted. Ethanol consumption resulted in dose-dependent reductions in osteocalcin, CTX, and the ratio of osteocalcin to CTX, indicative of reduced bone formation and resorption, as well as overall reduced bone turnover balance. Furthermore, while ethanol consumption did not result in the anticipated reduction in IGF-1 levels, evaluation of the relationships between IGF-1 and biochemical markers of bone turnover revealed a disruption of the normal positive relationships following the introduction of ethanol. This suggests that IGF-1 resistance may play a role in ethanol-induced alterations in bone turnover. Lastly, in contrast to the hypothesis, abstinence from ethanol consumption resulted in decreased osteocalcin and no change in IGF-1, and similar patterns were observed in the control animals. This unanticipated outcome may have been influenced by stress among the animals, as evidenced by elevated levels of cortisol which were associated with the altered levels of osteocalcin.