Very Low-Calorie Ketogenic Diet with Vegan Meal Replacements in an Obese Patient with Metabolic Syndrome Modifies Visceral Adipose Tissue Distribution and Taxonomic Composition of Gut Microbiota

: Short-term interventions based on very low-calorie ketogenic diets (VLCKDs) and meal replacements are a viable option for selected metabolic syndrome patients and/or to those with an altered microbiota composition. In this view, protein source choice is still debated. In fact, scarce and inconsistent data, are available on protein intake from various sources on body weight, composition of gut microbiota and metabolic outcomes in these patients. A 52-year-old man affected by first degree obesity, hypertension, hyperuricemia, hyperinsulinism, hypercholesterolemia, hypertriglyceridemia and altered gut microbiota with an increased Firmicutes/Bacteroidetes ratio, was admitted to our facility. Therefore, a nutritional treatment with VLCKD (≤800 kcal/day) was set for a 45 days period based on meal replacements containing vegetal proteins. Anthropometric indexes, blood and urine chemistry, body composition, muscle strength, taxonomic composition of the gut microbiome, were assessed at the beginning and after 45 days. A significant reduction of body weight (93 vs. 109 Kg), BMI (27 vs. 33 Kg/m 2) , blood pressure (120/70 vs. 150/100 vs. mmHg), waist circumference (102 vs. 114 cm), HOMA index (2,3 vs. 9,1), triglycerides (129 vs. 612 mg/dL), total cholesterol (151 vs. 318 mg/dL) and HDL cholesterol (48 vs. 32 mg/dl) and visceral adipose tissue (599 vs. 793 g) was observed at 0 – 45 days respectively. Importantly, a decrease in the relative abundance of Firmicutes, an increase in Bacteroidetes and Verrucomicrobiae abundance was also observed. These preliminary results suggest that the nutritional treatment with a vegan VLCKD led to a significant weight loss, a marked improvement of metabolic parameters and a restored eubiosis in gut microbiota. VLCKD with vegetal protein have promise as a valuable treatment for obesity, leading to an improvement of metabolic health, also related to a favourble rebalancing of gut microbiota.

Metabolic Syndrome (MetS) was described for the first time by Gerald Reaven in 1988 [1] and was defined as the association between insulin resistance (IR), hypertension, dyslipidemia, Nonalcoholic fatty liver disease (NAFLD), impaired glucose tolerance and other metabolic abnormalities related to increased cardiovascular risk [2].
Moreover MetS is linked to increase mortality for coronary heart disease (CHD), diabetes and cancer [3,4].
Although the pathophysiological mechanism is still under study, a key factor in the pathogenesis of MetS is represented by IR, a phenomenon prevalent in the obese population and characterized by the resistance of peripheral tissues to insulin action.According to the National Cholesterol Education Program's Adult Treatment Panel III report (NCEP ATP III) it is possible to diagnose metabolic syndrome when at least 3 of the following: Waist circumferences (WC) ≥ 102 cm man and ≥ 88 woman; Blood pressure (BP) ≥ 130/85 mmHg, or antihypertensive treatment; Triglycerides (Tg) ≥ 150 mg/dL or lipid lowering treatment; HDL < 39 mg/dL man <50 mg/dL woman; Glucose ≥ 101 mg/dL or hypoglycemic treatment [5].
Recent scientific evidence affirms that Very Low Calorie Ketogenic Diet (VLCKD) can be a valid treatment for patients with hyperinsulinism and visceral obesity [6][7][8].
It has been reported that the factors contributing to MetS are the result of extrinsic factors such as diet and lifestyle and complex host intrinsic factors such as genetics and gut microbiota [9].In particular it is known that in obese subjects there is a dysbiosis of gut microbiota [10,11]: obesity is characterized by an altered intestinal Bacteroides/Firmicutes ratio, with greater relative abundance of Firmicutes [12].This modification in gut microbiota composition is associated with greater energy breakdown and absorption from food, higher efficiency in storing fat in adipose tissue, as well as stimulation of metabolic endotoxemia and low-grade inflammation [9,13].
We hypothesized that VLCKD and subsequent modification of gut microbiota is strictly linked to the improvement of MetS.

Clinical Case
A 52-year-old man affected by first degree obesity, hypertension, hyperuricemia, hyperinsulinism, hypercholesterolemia, hypertriglyceridemia and altered gut microbiota with an increased Bacteroides/Firmicutes ratio, was admitted to our facility.Baseline medication therapy was carvedilol 6.25 mg, irbesartan 300 mg, hydrochlorothiazide 12.5 mg and allopurinol 300 mg.
This patient was screened from March 2019 to June 2019 and was prescribed VLCKD with meal replacements based on vegetal protein [780 kcal/day, with the following composition in macronutrients, percentage of caloric intake and g/kg of ideal BW of proteins (derived by the BMI set at 25 kg/m 2 ): carbohydrates 26 g (13.5%), olive oil 20 g plus 15 g of lipids from other sources (40.4%),proteins 90 g (46.1%, 1.4 g/Kg)] for 45 days.The amount of proteins was within the proposed essential composition of total diet replacements for weight control adjusted for the patients with obesity.
He ate five meals/day [timing was at main meals (8 a.m., 1.00 p.m. and 8.00 p.m.), mid-morning and mid-afternoon] containing vegetable protein derived from soya, green peas or cereals and one serving of vegetables with low glycemic index at lunch and dinner.Supplements of vitamins, minerals and omega-3 fatty acids, were provided in accordance to international recommendations (EFSA 2017).
Handgrip strength (HG) was measured through a digital dynamometer (DynEx, Akern, Pontassieve, FI, Italy) at T0 and T45.Three measurements were repeated with dominant and nondominant arms.The highest value measured was recorded.Body composition, total and regional body fat mass (FM), visceral adipose tissue (VAT), fat-free mass (FFM) and lean mass were measured through DEXA (Hologic 4500, Bedford, MA) at baseline and at the end.
Fecal sampling was done using sterile swab (FLmedical, Italy) and tubes (Starlab Group, Italy) in the morning of the day of starting VLCKD and at the end.
Following nutritional therapy, a significant reduction of body weight (93,4 vs. 109 Kg), BMI (27 vs. 33 Kg/m 2 ), waist circumference (102 vs. 114 cm) and blood pressure (120/70 vs. 150/100) was observed after 45 days (Table 1); the improvement of metabolic parameters led to a reduction of hypertension therapy (reduction of carvedilol and interruption of hydrochlorothiazide).Despite the change in therapy, blood pressure was normal and stable over time.Also, a reduction of HOMA index (2,3 vs. 9,1), total cholesterol (151 vs. 318 mg/dL), triglycerides (129 vs. 612 mg/dL) was observed at 0-45 days respectively (Table 2).At baseline the value of 612 mg/dl was confirmed and causes of pancreatitis and related diseases were excluded.Was also observed an increase of HDL cholesterol (48 vs. 32 mg/dL).DEXA analysis showed a decrease of VAT (599 vs. 793 g) (Figure 1).Furthermore, a reduction in IGF-1 circulating levels (100 vs. 140 ng/mL) and an increase in SIRT (264 vs. 144 ng/mL) were observed after 45 days.Dominant phyla in fecal samples from the patient at T0 were Firmicutes, Bacteroidetes, Proteobacteria, Verrucomicrobia, Fusobacteria and Actinobacteria.Relative abundance of Firmicutes was significantly reduced and Bacteroidetes proportionally increased 45 days after the beginning of VLCKD.The mean relative abundance of Proteobacteria also increased while Actinobacteria decreased (not shown).An increase of Verrucomicrobia was also observed (Figure 2).
This case report shows that VLCKD caused a marked reduction of BW (in the range of 15%), leading the patients from grade I obesity to overweight.It also improves parameters of metabolic syndrome with a subsequent reduction of cardiovascular risk.
An important concern with low carbohydrate diets relies on the potential negative impact on lipid metabolism due to the increased proportion of calories resulting from fat.Clearly, this does not apply to a 45 days VLCKD, for which the daily lipids intake is low as can be inferred by the profound reduction of the circulating cholesterol and triglycerides observed in our patient.Many other mechanisms may contribute to the reduction of circulating lipids; the improvement of insulin resistance with the positive effects on lipid metabolism through the action on HMG-CoA reductase and the striking effects on lipoprotein size and subclass particle concentrations [18].
IGF-1 decreased is related to reduced risk of all cancers, as already documented [19][20][21].To date, studies suggest limiting protein consumption to achieve a reduction.From the results that emerge from our case, it would therefore not seem necessary to reduce or even eliminate proteins from dietary therapy [22], but it is necessary to use vegetable proteins.
The increase in SIRT1 is determined by weight loss [23,24].SIRT1 is an antioxidant factor that causes further protection for T2DM and NAFLD [25].
Body composition changed with a marked reduction in VAT, which is directly correlated with the reduction of cardiovascular risk.Lean mass was preserved with subsequent preservation of muscle efficiency, thanks to the correct amount of protein (1.4 g/kg).
The VLCKD was safe and no majorn side effects were reported.An increase in serum acid uric was observed (7,7 vs. 5.5 μmol/L), despite therapy.As already found in the literature, this increase is due to the increase in soy content, present in meal replacements [26].
The gut microbiota composition is strictly influenced by the VLCKD and the source of dietary proteins modulates the variation of gut microbiota.In only 45 days we observed a reduction in Firmicutes, which are usually particularly abundant in obese subjects and are correlated with chronic low-grade inflammation, which supports the pathophysiological mechanisms underlying the mechanisms of insulin resistance.The most interesting aspect is the increase in Verrucomicrobia: Akkermansia muciniphila is the main genus of this phylum in human gut.An increase in Akkermansia m. has been shown to lead to an improvement in barrier function, endotoxemia and insulin sensitivity, which could lead to protection from T2DM and all other MetS cofactors [27,28].Further studies are needed to better clarify this increase and the role of this bacterium.

Conclusions
In conclusion, our report indicates that VLCKDs may hold promise as a strategy to improves parameters of metabolic syndrome, allowing a profound weight loss in patients with insulin resistance, obesity and MetS.This leads to a reduction in cardiovascular risk in only 45 days.This is the first case-report showing a favourable effect of a VLCKD with veg proteins in gut microbiota.
The improvement of the Bacteroides/Firmicutes ratio and the increase in Verrucomicrobia could represent additional determining factors for choosing a VLCKD with vegan meal replacement in this type of patient.

Figure 1 .
Figure 1.Measurement of VAT by DXA at baseline (Panel A) and after 45 days (Panel B).

Figure 2 .
Figure 2. Effect of VLCKD dietary interventions with vegetable protein on the relative abundance of Firmicutes, Bacteroidetes and Verrucomicrobia at baseline (A) and after 45 days (B).

Table 1 .
Participant characteristics of anthropometric measurements at baseline (T0) and every two weeks of VLCKDs.

Table 2 .
Characteristics of blood test and body composition parameters at baseline (T0) and after 45 days (T 45) of VLCKDs.