This was a single-arm pilot trial of an online, VLC dietary intervention.

The research was approved by the University of Michigan Institutional Review Board (HUM00154361) and registered at ClinicalTrials.gov (NCT05010070). The study followed the ethical standards of the responsible committee on human experimentation (institutional and national) and abided by the Helsinki Declaration of 1975, as revised in 2000. All participants provided informed consent and could withdraw from the study at any point. No writing assistance was used for this manuscript. Recruitment began on August 31, 2021, and the outcomes of the trial were finalized by October 3, 2022.

Potential participants were contacted based on their rs738409 genotype in the Michigan Genomics Initiative (MGI), a research initiative that collects and genotypes blood samples and allows researchers to link this genetic information to patients. As part of the initiative, participants had already provided informed consent for broad research purposes [24]. Participants interested in our pilot study were referred to a webpage that described the trial and linked to an online screening survey (Qualtrics). The screening survey included questions used to assess eligibility. If individuals passed this, they were sent a video describing the goals, procedure, and pros and cons of participating in the trial. If they continued to express interest in the trial, they consented, baseline measures were collected, and then they were enrolled in the trial. Inclusion criteria included a baseline magnetic resonance imaging with liver steatosis but not cirrhosis, having been identified based on information from the MGI database, and having elevated liver function tests. Exclusion criteria included non-MASLD causes of elevated liver function tests; use of exogenous insulin; planned or history of weight loss surgery; active substance use or untreated mental health condition that could pose a safety risk; advanced medical conditions including as current chemotherapy, heart failure, or kidney failure; type 1 diabetes; Cushing syndrome; adherence to a vegan or dietary vegetarian; pregnant or planning to get pregnant in the next 6 months; currently enrolled in a weight loss program or other investigative study that might conflict with this research; taking medications known to cause weight gain or loss; or recent decompensation/hospitalization.

The online intervention included 16 weekly emails with a video and handouts, as well as text messages and email-based coaching. At baseline, participants received a body weight scale (Bodytrace) and urine ketone strips. Participants were also sent VLC cookbooks by mail 3 times throughout the intervention [25-27]. We taught participants to follow a VLC eating pattern, consisting of 20‐35 net (nonfiber) grams of carbohydrates per day. At week 6, we encouraged participants to become physically active for at least 150 minutes of moderate-intensity physical activity per week as well as to sleep enough to feel well rested. The intervention also taught skills related to feeling more positive affect and eating mindfully [28]. Throughout, we asked participants to track their dietary intake and to step on the body weight scale at least once a week. The program’s email-based coach provided feedback and support and we sent near daily, unique text messages that provided reminders of the intervention content.

At baseline, participants were asked to self-weigh using the scale, complete a fasting blood draw and magnetic resonance imaging at Michigan Medicine, complete an online survey (Qualtrics); and complete a 2-day dietary recall with a trained dietitian by phone. At 4 months, at the end of the intervention, these were repeated. Prior to each week’s didactic session, we asked participants questions about their dietary adherence and experiences. Procedures associated with the research were either covered by the study or the participants’ insurance.

For statistical significance, we followed the guidance for the use of frequentist inferential statistics in public health [35]. We completed an intention-to-treat analysis with all available data and analyses that were limited to only adherent individuals, with no imputation for missing data. Means and 95% CIs were calculated for continuous variables.

Of the 97 individuals who expressed interest in study participation and were screened for study eligibility, 55 did not meet inclusion criteria (including, as the top reasons for ineligibility: 13 were taking insulin, 11 had prior weight loss surgery or were taking weight loss medications, 8 had normal liver function tests, 6 had cirrhosis of the liver, 3 had heart failure or another related heart condition), 12 were eligible based on the prescreening survey but did not reply to follow-up requests, and 5 were eligible but declined to participate. Eleven participants enrolled in the trial.

At baseline, participants were 38‐77 years old (mean 55.54 years, SD 11.85 years), 8 out of 11 (72.7%) participants were male, most were White and not Hispanic (10/11, 90.9%), with 1 being White and Hispanic. Of the 11 participants, 7 (63.6%) participants were rs738409-GG and 4 (36.4%) participants were rs738409-CG. Other baseline values are in Table 1.

Results suggest the feasibility, acceptability, and preliminary efficacy of the VLC intervention in adults with higher genetic risk for MASLD, although there is a need for further studies given the small sample size and the high risk of substantial biases in this small pilot study. One concern of the VLC diet is its tolerability. Here we show that the diet was well tolerated with 5 out of 9 participants rating it at the highest satisfaction score. Only 1 out of 9 said they would stop the assigned dietary pattern as soon as the study was over. In contrast, 4 out of 9 said they would continue to follow the diet indefinitely. This is consistent with data where close to 60% of individuals with relapsing multiple sclerosis continued on a VLC or low-carb diet following a trial of the diet, particularly if they had lost a lot of weight and had decreased fatigue [36]. The mindfulness training was less helpful to participants with only 1 out of 9 rating it with the top score. They noted challenges with adhering to the diet as time, carbohydrate cravings, fatigue, vacations, and social situations which need to be addressed as part of a larger program to change eating patterns.

Several previous trials have examined the impact of a VLC eating pattern for adults with MASLD or metabolic dysfunction–associated steatohepatitis, and they found similar results. For example, in a 1-month trial of a VLC diet with 27 participants with MASLD, left hepatic lobe volume decreased by 20% [37]. In a 3-month trial of a Spanish and Mediterranean–adapted VLC diet in 12 men, liver steatosis improved in 93% of participants, and complete fatty liver regression was observed in 21% of participants [12]. In a 6-month trial of a VLC diet with 5 participants, 4 (80%) showed improvements in liver histology, steatosis, inflammatory grade, and fibrosis [10]. In a 2-month trial of a very low-calorie, VLC diet with 20 participants with obesity, the prevalence of liver steatosis (proton density fat fraction>5.4%) decreased from 70% to 30%. Likewise, 2 other nonrandomized, pre-post trials of hypocaloric VLC diets, a 6.5-month [38] and an 8-week trial, both showed improvements in liver steatosis [39].

Five previous trials, with a total of 179 people, have randomized with MASLD to a Mediterranean diet and measured liver-based fat with magnetic resonance imaging. These varied in length of time, with 1 being 1.5 months long, 2 being 3 months long, and 3 being 6 months long. Overall, they have found an average of 12%‐60% relative reduction in hepatic fat percentage [40-44]. Our results and the results of previous VLC trials suggest that a VLC, ketogenic diet may be at least as effective as a Mediterranean diet, although future randomized trials are needed to assess this. Our study is also in line with a previous study where carriers of PNPLA3 G risk allele were able to lose substantial liver fat on a low carbohydrate diet [45].

One of our major limitations is the fact that our sample size was small. Our recruitment was limited by the fact that we reached out to adults who had already been genotyped by the MGI. Instead, if we had genotyped interested participants, it may have been easier to recruit more participants. We also required that some of the study-related tests, which could be justified as medical expenses as part of regular health care, including the baseline magnetic resonance imaging and most of the blood tests, be covered by the participants’ health insurance. However, not all potential participants were willing to add these charges to their insurance, because it would still incur costs to the participants. In addition, the trial was conducted during the COVID-19 pandemic, which may have influenced who was willing or able to participate or their ability to adhere to dietary instructions.

Moreover, this trial’s generalizability is limited, as most participants were White, non-Hispanic men. Future recruitment efforts should focus on recruiting a more diverse population. This trial lacked a control group to which participants could have been randomized to, and thus statements cannot be made about causality.

Another important limitation is duration. As with other trials in this area, participants were tracked for less than a year, which does not allow for the understanding of the longer-term impacts of this nutritional approach nor whether participants may maintain dietary adherence longer term [10,12,37-44]. A growing body of literature including our paper showed that both insulin resistance and the PNPLA3 risk allele can drive the progression of liver disease [46,47]. A limitation of our study is that whether PNPLA3-driven liver fat accumulation causes liver insulin resistance cannot be determined here. Finally, the lack of a PNPLA3 CC control group limits our ability to determine whether the outcomes would be even more improved in the G versus C carriers.

Thus, results supported the feasibility, acceptability, and preliminary efficacy of the VLC intervention in adults thought to have a higher genetic risk for MASLD. Overall, there is a need for longer-term studies to assess the sustainability of benefits and potential long-term risks associated with this way of eating. Moreover, future studies should aim to include a more diverse population, explore the biological mechanisms by which the VLC diet affects MASLD, consider the role of the PNPLA3 genotype, and integrate omics technologies to identify modulated biomarkers and pathways, thereby enhancing the generalizability and depth of the findings.