Experience with dronabinol consumption facilitated a stimulant effect of alcohol and affected alcohol-related changes in frontal cortical endocannabinoid levels in male rats.

Combined use of cannabis and alcohol is common in adolescents. However, the extent to which such polydrug exposure affects the brain and behaviors remains under-investigated in preclinical studies. This study tested the hypothesis that combined exposure of Δ-9-tetrahydrocannabinol (THC), the main psychoactive constituent of cannabis, and alcohol will have additive effects on cognitive impairments and altered endocannabinoid levels in the hippocampus and frontal cortex. Male Long Evans rats were provided with daily access to cookies laced with oil or dronabinol, a synthetic THC, during adolescence. Three days after discontinuation of edible THC, the effect of orally administered 3 g/kg alcohol on Barnes maze performance was assessed. The results showed that experience with edible THC facilitated the occurrence of increased moving speed on the maze induced by repeated alcohol administration. However, contrasting to the hypothesis, the combined THC and alcohol exposure did not lead to additive deficits in learning and memory on the Barnes maze. While little effect on endocannabinoid levels was observed in the hippocampus, acute abstinence from alcohol significantly reduced endocannabinoid levels in the frontal cortex. In particular, reduction of N-oleoyl ethanolamine (OEA) and N-stearoyl ethanolamine (SEA) were robust and had an interactive effect with discontinuation from edible THC. These findings add to the scarce literature on THC and alcohol associated changes in endocannabinoid levels and provide insights to future investigations on the roles of OEA and SEA on physiology and behaviors following THC and alcohol co-exposure during adolescence.

A gene-diet interaction controlling relative intake of dietary carbohydrates and fats.

OBJECTIVE: Preference for dietary fat vs. carbohydrate varies markedly across free-living individuals. It is recognized that food choice is under genetic and physiological regulation, and that the central melanocortin system is involved. However, how genetic and dietary factors interact to regulate relative macronutrient intake is not well understood. METHODS: We investigated how the choice for food rich in carbohydrate vs. fat is influenced by dietary cholesterol availability and agouti-related protein (AGRP), the orexigenic component of the central melanocortin system. We assessed how macronutrient intake and different metabolic parameters correlate with plasma AGRP in a cohort of obese humans. We also examined how both dietary cholesterol levels and inhibiting de novo cholesterol synthesis affect carbohydrate and fat intake in mice, and how dietary cholesterol deficiency during the postnatal period impacts macronutrient intake patterns in adulthood. RESULTS: In obese human subjects, plasma levels of AGRP correlated inversely with consumption of carbohydrates over fats. Moreover, AgRP-deficient mice preferred to consume more calories from carbohydrates than fats, more so when each diet lacked cholesterol. Intriguingly, inhibiting cholesterol biosynthesis (simvastatin) promoted carbohydrate intake at the expense of fat without altering total caloric consumption, an effect that was remarkably absent in AgRP-deficient mice. Finally, feeding lactating C57BL/6 dams and pups a cholesterol-free diet prior to weaning led the offspring to prefer fats over carbohydrates as adults, indicating that altered cholesterol metabolism early in life programs adaptive changes to macronutrient intake. CONCLUSIONS: Together, our study illustrates a specific gene-diet interaction in modulating food choice.

Joint and separate exposure to alcohol and ∆9-tetrahydrocannabinol produced distinct effects on glucose and insulin homeostasis in male rats.

Cannabis and alcohol co-use is common, and the trend may increase further given the current popularity of cannabis legalization. However, the metabolic consequences of such co-use are unclear. Here, we investigated how co-administration of alcohol and ∆9-tetrahydrocannabinol (THC), the main psychoactive constituent of cannabis, affects body weight and visceral adiposity, and glucose and insulin homeostasis in rats. For 16 consecutive days during adolescence, male rats drank saccharin or alcohol after receiving subcutaneous oil or THC injections in Experiment 1 and voluntarily consumed alcohol, THC edible, or both drugs in Experiment 2. Experiment 1 showed that following abstinence, drug co-exposure reduced visceral fat and the amount of insulin required to clear glucose during an oral glucose tolerance test (OGTT). In Experiment 2, rats received a high-fat diet (HFD) after 3-week abstinence. Although adolescent drug use did not interact with the HFD to worsen hyperglycemia and hyperinsulinemia during an OGTT, HFD-fed rats that co-used alcohol and THC had the lowest insulin levels 75 min after an insulin injection, suggesting an altered rate of insulin secretion and degradation. These results suggest that THC and alcohol co-exposure can distinctly alter the physiology of glucose and insulin homeostasis in a rodent model.

Combined ∆9-tetrahydrocannabinol and moderate alcohol administration: effects on ingestive behaviors in adolescent male rats.

RATIONALE: Whereas co-use of alcohol and marijuana is prevalent in adolescents, the effects of such drug co-exposure on ingestive and cognitive behaviors remain largely unexplored. We hypothesized that co-exposure to alcohol and ∆9-tetrahydrocannabinol (THC), the main psychoactive constitute of marijuana, alters feeding behavior and cognition differently from either drug alone. METHODS: Male rats received daily THC (3-20 mg/kg/day) or oil vehicle through subcutaneous injection or consumption of a cookie with access to saccharin or saccharin-sweetened alcohol during adolescence (P30-45). Barnes maze and sucrose preference tests were applied to assess spatial memory and behavioral flexibility and abstinence-related anhedonia, respectively. RESULTS: Subcutaneous THC did not affect alcohol intake but dose-dependently increased acute (3 h) chow intake and reduced weight gain. Moderate alcohol consumption reduced the acute hyperphagic effect of subcutaneous THC. By contrast, oral THC at a dose > 5 mg/kg robustly reduced alcohol intake without affecting 3-h chow intake. At this dose, some rats stopped consuming the THC-laced cookies. Furthermore, oral THC reduced weight gain, and co-exposure to alcohol alleviated this effect. Chronic subcutaneous, but not oral, THC reduced sucrose intake during abstinence. Neither treatment impaired cognitive behaviors in the Barnes maze. CONCLUSION: Moderate alcohol and THC consumption can interact to elicit unique outcomes on ingestive behaviors and energy balance. Importantly, this study established a novel model of voluntary alcohol and THC consumption for studying mechanisms underlying the consequences of adolescent onset co-use of the two drugs.

Chronic moderate alcohol drinking alters insulin release without affecting cognitive and emotion-like behaviors in rats.

Because the consumption of alcoholic beverages prevails in society, its effects on diabetes risk is a subject of interest. Extant literature on this issue often disagrees. Here, we probed the effects of chronic moderate ethanol consumption on glucose metabolism in rats. The effect of chronic moderate alcohol drinking on depression- and anxiety-like behaviors and memory was also explored. Adolescent male and female Long-Evans rats consumed saccharin-sweetened 5% (1 week) and 10% ethanol (7 weeks) under a 7.5-h/day (Monday-Friday) access schedule. This exposure was followed by sucrose preference and elevated plus maze (EPM) tests during an intervening week, before a 6-week intermittent-access (Monday, Wednesday, Friday) to 20% unsweetened ethanol in a 2-bottle choice drinking paradigm was implemented (EtOH). A free-feeding control group received water (Water). Our prior work revealed that voluntary ethanol consumption decreases food intake in rats. Hence, a second control group that received water was mildly food-restricted (FR), and their average body weight was matched to that of the EtOH group. During the week following week 6 of intermittent-access to 20% ethanol, rats were submitted to sucrose preference, EPM, and novel object recognition (NOR) tests. Insulin response to a glucose load was subsequently assessed via an oral glucose tolerance test (OGTT). Rats attained and maintained blood ethanol concentrations of ∼55 mg/dL that correlated with the dose of sweetened 10% ethanol ingested. Relative to intake by Water controls, EtOH rats consumed less chow. There was no body weight difference between both groups. Neither sex of EtOH rats showed increased depression- and anxiety-like behaviors, as respectively measured by sucrose preference and EPM, nor did they show deficit in object recognition memory during abstinence. Male EtOH rats, however, showed signs of reduced general activity on the EPM. During OGTT, male EtOH rats showed a time-dependent potentiation of insulin release for proper glucose clearance. Such an effect was not observed in females. This landmark study shows that chronic moderate alcohol consumption can have negative metabolic consequences in the absence of overt behavioral deficits, especially in males.

Appetite and weight gain suppression effects of alcohol depend on the route and pattern of administration in Long Evans rats.

Ethanol can be a food source but its effects on energy balance and contribution to obesity remain inconclusive. In this study, we hypothesized that the effects of ethanol on energy intake and body weight would depend on the administration dose, pattern and the blood ethanol concentration (BEC) time-course. Experiment 1 examined changes in food intake, diet preference, and body weight after saline or ethanol (1 and 3g/kg) injection (IP). Experiment 2 compared the effects in rats that received either 3g/kg/day ethanol administered all at once (EtOH_S) or 2 1.5g/kg injections spaced by 3h (EtOH_D). Experiment 3 examined the effects of 7.5h/day, Mon through Fri for 8weeks, voluntary ethanol drinking (5% and 10% ethanol) on food intake and body weight. Results of Experiments 1 and 2 indicate that acute ethanol administrations dose-dependently reduced energy intake, high fat diet preference and weight gain. Acute 3g/kg ethanol injection in the EtOH_S group decreased energy intake, weight gain and visceral fat to a greater extent than in the EtOH_D group. Results of Experiment 3 show that male and female rats voluntarily drank 1.65-2.31g/kg ethanol within 3.5h with reduced chow intake but unchanged total energy intake and weight gain. Furthermore, 3g/kg ethanol injection resulted in BEC that remained at intoxicating levels e.g. >120mg/dL for several hours post-administration and was higher in the EtOH_S than in the EtOH_D group. In contrast, BEC in voluntarily drinking was ~67mg/dL and decreased to below 10mg/dL 5h after termination of ethanol access. Taken together, these data suggest that 3g/kg ethanol injection robustly suppresses appetite and weight gain due to the higher BECs attained. Furthermore, BEC attained and maintained is a determining factor for how ethanol administration affects appetite and long-term energy balance.

CAPN5 gene silencing by short hairpin RNA interference.

BACKGROUND: The purpose of this project was to identify short hairpin RNA (shRNA) sequences that can suppress expression of human CAPN5 in which gain-of-function mutants cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). We created HEK293T cells that stably express an ADNIV disease allele, CAPN5-p.R243L. Transfection protocols were optimized for neuroblastoma SHSY5Y cells. The gene silencing effect of four different shRNA plasmids that target CAPN5 was tested. RNA and protein expression was determined using quantitative RT-PCR and immunoblot analysis. FINDINGS: Two of four shRNA plasmids reduced mutant CAPN5 RNA in a stable cell line. Similar knockdown was observed in SH-SY5Y cells that natively express CAPN5. Lactose dehydrogenase assays showed that down-regulation of CAPN5 was not cytotoxic. CONCLUSIONS: CAPN5 expression can be suppressed by shRNA-based RNA interference. Further testing in ADNIV models will determine the potential of gene silencing as a strategy to treat, delay, or prevent blindness in ADNIV patients.